Nucleic acids encoding agonistic antibodies that bind CD40

ABSTRACT

Isolated monoclonal agonistic antibodies which bind to human CD40 and related antibody-based compositions and molecules are disclosed. Also disclosed are therapeutic and diagnostic methods for using the antibodies.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/324,170, filed Apr. 18, 2016. The contents of the aforementionedapplication is hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 6, 2017, isnamed Sequence-Listing_CDJ-393.txt and is 75,696 bytes in size.

BACKGROUND OF THE INVENTION

Interactions between T cells and antigen-presenting cells involve avariety of accessory molecules that facilitate the generation of animmune response. One such molecule is CD40, a member of the tumornecrosis factor receptor (TNF-R) superfamily which binds to CD40L(Ranheim E A, et al., Blood. 1995 Jun. 15; 85(12):3556-65). CD40 is atransmembrane 43-48 kDa glycoprotein composed of 277 amino acid residues(Braesch-Andersen et al., 1989). CD40 is expressed by antigen-presentingcells (APC) and engagement of its natural ligand (CD40L) on T cellsactivates APC including dendritic cells and B cells (Khalil andVonderhide (2007) Update Cancer Ther, 2(2): 61-65), thus enhancingimmune responses. CD40 is also expressed on many tumor cells and itsligation in this setting mediates a direct cytotoxic effect, e.g.,engagement of CD40 on tumor cells results in apoptosis in vitro andimpaired tumor growth in vivo (Tai et al. (2004) Cancer Res,64(8):2846-52).

Monoclonal antibodies against CD40 provide a variety of potentialtherapeutic purposes including the treatment of cancers. For example,agonistic CD40 antibodies have been shown to substitute for T cell helpprovided by CD4+ lymphocytes in murine models of T cell-mediatedimmunity, and in tumor-bearing hosts CD40 agonists trigger effectiveimmune responses against tumor-associated antigens (Bennett et al.(1998) Nature, 393(6684):478-80). In addition, CD40 antibodies holdgreat promise for use in vaccines (Fransen et al. (2014) Vaccine32:1654-1660). However, there are potential adverse effects associatedwith agents that strongly modulate the immune system (Sandin et al.(2014) Cancer Immunol Res, 2:80-90). Accordingly, there is a need forfurther insight into the specific properties and mechanisms that makeCD40 antibodies therapeutically effective, as well as improvedtherapeutic antibodies against CD40 that can be used to treat and/orpreventing diseases.

SUMMARY OF THE INVENTION

The present invention provides isolated anti-CD40 antibodies havingparticular functional properties which can be linked with advantageousand desirable therapeutic effects. Specifically, agonistic anti-CD40monoclonal antibodies capable of increasing an immune response to anantigen (e.g., an antigen expressed on a cell) have been generated andcharacterized. As used herein, the term “antibody” refers to full-lengthantibodies and antigen binding portions thereof.

In one embodiment, the anti-CD40 antibodies enhance immune responsesagainst an antigen, e.g., by enhancing T cell-mediated immune responses,B-cell activation, and/or cytokine production. The antibodies can beadministered alone or in combination therapies (e.g., with vaccinetherapy and/or chemotherapy).

In another embodiment, the anti-CD40 antibodies are capable ofincreasing an immune response to an antigen without inducingantibody-dependent cellular cytotoxicity (ADCC) of CD40 expressing cellsand/or complement dependent cellular cytotoxicity (CDC) of CD40expressing cells.

In another embodiment, the antibodies comprise an effectorless constantregion. In one embodiment, the constant region is an IgG2 isotype (e.g.,human IgG2).

In yet another embodiment, the anti-CD40 antibodies exhibit one or moreof the following properties:

(a) no blocking of binding of CD40L to human CD40, independent of Fcreceptor binding;

(b) blocking of binding of CD40L to human CD40, independent of Fcreceptor binding;

(c) activation of human CD40 expressed on an Antigen Presenting Cell(APC), independent of Fc receptor binding;

(d) induction of apoptisis of a tunore cell;

(e) T-cell stimulatory activity;

(f) enhanced B-cell activation; and/or

(g) capable of synergising with CD40L.

Preferably the antibodies act independently of Fc receptor interaction.Preferably the antibodies are IgG2 isotype antibodies.

In one embodiment, the agonistic antibodies are capable of increasing animmune response independent of Fc receptor binding. For example, theantibodies may exhibit potent agonistic features without cross-linkingwith an Fc receptor, such as FcγR. These agonistic features include,e.g., an increase in T-cell activity and/or an increase in B cellactivation as measured, e.g., by an increase in the expression of cellsurface markers selected from the group consisting of HLA-DR V450, CD54PE, CD86 APC, CD83 BV510, CD19 V500, CD54 PE, HLA-DR V450, CD23PerCP-Cy5.5, CD69 APC, CD86 APC, CD38 and CD71 PE.

In another embodiment, the antibodies block binding of CD40 to CD40L(CD154) on CD40-expressing cells. In particular embodiments, theantibodies inhibit the binding of soluble CD40L to CD40 expressing cellsby at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In aparticular embodiment, the anti-CD40 antibody inhibits binding of CD40Lby at least about 70% as measured, e.g., by FACS, bio-layerinterferometry (BLI) or Biacore. In another embodiment, the anti-CD40antibody inhibits binding of CD40L by at least about 80% as measured bye.g., by FACS, BLI or Biacore.

In another embodiment, the antibodies induce apoptosis of cells, asmeasured, e.g., by increased expression of CD95. The antibodies also canbe constructed to include an Fc region which has specificity for aparticular Fc receptor (e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB1(CD32), FcγRIIB2 (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), FcεRI,FcεRII (CD23), FcαRI (CD89), Fcα/μR, and FcRn).

In another embodiment, the antibodies are capable of binding to humanCD40 with an equilibrium dissociation constant Kd of 10⁻¹⁰ M or less,preferably 10⁻¹¹ M or less and/or cross-reacting with cynomolgus CD40.

Particular anti-CD40 antibodies of the invention include antibodies 3C3,3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, 3B6-NS, and related embodimentsdescribed below.

In one embodiment, the antibodies comprise a heavy chain variable regionCDR3 sequence selected from the group consisting of SEQ ID NOs: 9, 10,23, 24, 37, 38, 51, 52, 65, 66, 65, 66, 79, 80, 93, 94, 107, 108,including conservative sequence modifications thereof (e.g.,conservative amino acid substitutions). The antibodies may furthercomprise light chain variable region CDR3 sequence selected from thegroup consisting of SEQ ID NOs: 15, 16, 29, 30, 43, 44, 57, 58, 71, 72,85, 86, 99, 100, 113, 114, including conservative sequence modificationsthereof. In another embodiment, the heavy chain CDR2 and/or CDR1sequences are selected from SEQ ID NOs: 7, 8, 21, 22, 35, 36, 49, 50,63, 64, 77, 78, 91, 92, 105, 106, and SEQ ID NOs: 5, 6, 19, 20, 33, 34,47, 48, 61, 62, 61, 62, 75, 76, 89, 90, 103, 104, respectively,including conservative sequence modifications thereof. In anotherembodiment, the light chain CDR2 and/or CDR1 sequences are selected fromSEQ ID NOs: 13, 14, 27, 28, 41, 42, 55, 56, 69, 70, 84, 85, 97, 98, 111,112, and SEQ ID NOs: 11, 12, 25, 26, 40, 41, 53, 54, 67, 68, 81, 82, 95,96, 109, 110, respectively, including conservative sequencemodifications thereof.

In another embodiment, the antibodies comprise a heavy chain variableregion having an amino acid sequence selected from the group consistingof SEQ ID NOs: 3, 17, 31, 45, 59, 73, 87, 101, including conservativesequence modifications thereof. The antibodies may further comprise alight chain variable region having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 4, 18, 32, 46, 60, 74, 88, 102,including conservative sequence modifications thereof.

In another embodiment, antibodies comprise heavy and/or light chainvariable regions respectively having the following amino acid sequences(including conservative sequence modifications):

(a) SEQ ID NOs: 3 and/or 4;

(b) SEQ ID NOs: 17 and/or 18;

(c) SEQ ID NOs: 31 and/or 32;

(d) SEQ ID NOs: 45 and/or 46;

(e) SEQ ID NOs: 59 and/or 60;

(f) SEQ ID NO: 73 and/or 74;

(g) SEQ ID NO: 87 and/or 88; or

(h) SEQ ID NO: 101 and/or 102.

Antibodies which include heavy and light chain variable regions havingat least 80%, or at least 85%, or at least 90%, or at least 95%, or atleast 96%, or at least 97%, or at least 98%, or at least 99%, or greatersequence identity to any of the above sequences also are included in thepresent invention. Ranges intermediate to the above-recited values,e.g., heavy and light chain variable regions having at least 80-85%,85-90%, 90-95% or 95-100% sequence identity to any of the abovesequences also are encompassed by the present invention.

In yet another embodiment, the antibodies bind to human CD40 and havethe CDR sequences from the heavy and light chain variable regionsrespectively having the amino acid sequences as set forth in:

(a) SEQ ID NOs: 3 and 4;

(b) SEQ ID NOs: 17 and 18;

(c) SEQ ID NOs: 31 and 32;

(d) SEQ ID NOs: 45 and 46;

(e) SEQ ID NOs: 59 and 60; or

(f) SEQ ID NO: 73 and 74;

(g) SEQ ID NO: 87 and 88; or

(h) SEQ ID NO: 101 and 102

(in each case including one conservative sequence modification, twoconservative sequence modifications, or up to three, up to four, or upto five conservative sequence modifications within one or more CDRs).

In another embodiment, the antibodies binds to human CD40 and have:

(a) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 5,7, 9, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 11, 13, 15, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:19, 21, 23, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 25, 27, 29, respectively, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:33, 35, 37, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 39, 41, 43, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:47, 49, 51, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 53, 55, 57, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:61, 63, 65, respectively and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 67, 69, 71, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:75, 77, 79, respectively and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 81, 83, 85, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:89, 91, 93, respectively and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 95, 97, 99, respectively; or

(h) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:103, 105, 107, respectively and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 109, 111, 113, respectively, (in eachcase optionally including one conservative sequence modification, twoconservative sequence modifications, or up to three, up to four, or upto five conservative sequence modifications within one or more of saidCDRs).

In yet another embodiment, the antibodies binds to human CD40 and have:

(a) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 6,8, 10, respectively, and/or light chain CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 12, 14, 16, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:20, 22, 24, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 26, 28, 30, respectively, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:34, 36, 38, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 40, 42, 44, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:48, 50, 52, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 54, 56, 58, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:62, 64, 66, respectively and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 68, 70, 72, respectively; or

(f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:76, 78, 80, respectively and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 82, 84, 86, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:90, 92, 94, respectively and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 96, 98, 100, respectively; or

(h) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:104, 106, 108, respectively and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 110, 112, 114, respectively, (in eachcase optionally including one conservative sequence modification, twoconservative sequence modifications, or up to three, up to four, or upto five conservative sequence modifications within one or more of saidCDRs).

In another aspect, the invention provides antibodies which compete forbinding to CD40 with the particular antibodies described above. In oneembodiment, the antibody competes for binding to CD40 with an antibodycomprising heavy and/or light chain variable regions comprising theamino acid sequences set forth in SEQ ID NOs: 3 and 4, SEQ ID NOs: 17and 18, SEQ ID NOs: 31 and 32, SEQ ID NOs: 45 and 46, SEQ ID NOs: 59 and60, SEQ ID NOs: 73 and 74, SEQ ID NO: 87 and 88, SEQ ID NO: 101 and 102,respectively.

In another aspect, the invention provides antibodies that bind to thesame epitope as, or an epitope on CD40 recognized by, the particularantibodies described above. In one embodiment, the antibody binds to anepitope on CD40 recognized by an antibody comprising heavy and/or lightchain variable regions comprising the amino acid sequences set forth inSEQ ID NOs: 3 and 4, SEQ ID NOs: 17 and 18, SEQ ID NOs: 31 and 32, SEQID NOs: 45 and 46, SEQ ID NOs: 59 and 60, SEQ ID NOs: 73 and 74, SEQ IDNO: 87 and 88, SEQ ID NO: 101 and 102, respectively. In someembodiments, the antibody binds to the same epitope as antibody 3C3 or3G5.

In another aspect, the invention provides antibodies that bind to one ormore residues within amino acid residues 1-5 and 33-36 of theextracellular domain (ECD) of human CD40 (SEQ ID NO: 133). In someembodiments, the antibodies further bind to one or more amino acidselected from the group consisting of amino acids 25, 26, 28 and 30 ofthe ECD of human CD40 (SEQ ID NO: 133). In some embodiments, theantibodies bind to one or more amino acids selected from the groupconsisting of amino acids 5, 33, 34 and 36 of the ECD of human CD40 (SEQID NO: 133). In some embodiments, the antibodies bind to amino acids 5,33 and 36 of the ECD of human CD40 (SEQ ID NO: 133). In someembodiments, the antibodies bind to amino acids 5, 33, 34 and 36 of theECD of human CD40 (SEQ ID NO: 133).

In any of the foregoing aspects, the invention provides antibodieswherein substitution of alanine with threonine at position 5 of the ECDof human CD40 (SEQ ID NO: 133) reduce binding of the antibodies by atleast 30% relative to bind to the ECD of human CD40 (SEQ ID NO: 133). Insome embodiments, substitution of alanine with threonine at position 5of the ECD of human CD40 reduces binding of the antibodies by at least50% relative to binding to the ECD of human CD40 (SEQ ID NO: 133). Insome embodiments, substitution of alanine with threonine at position 5of the ECD of human CD40 reduces binding of the antibodies by at least80% relative to binding to the ECD of human CD40 (SEQ ID NO: 133).

In any of the foregoing aspects, the invention provides antibodies thatexhibit a synergistic effect with CD40L which may be endogenous CD40L.In some embodiments, the synergistic effect is increased induction ofCD95 expression when incubated with Ramos cells. In some embodiments,the synergistic effect is an increase in B cell proliferation whenincubated with human B cells. In some embodiments, the synergisticeffect is increased induction of IL12p40 expression when incubated withdendritic cells. In some embodiments, the synergistic effect is measuredin terms of expression of CD95.

In another aspect, the invention provides antibodies that bind to one ormore residues within amino acid residues 13-15 and 33-36 of the ECD ofhuman CD40 (SEQ ID NO: 133). In some embodiments, the antibodies bind toone or more amino acids selected from the group consisting of aminoacids 33, 34 and 36 of the ECD of human CD40 (SEQ ID NO: 133).

Antibodies of the invention can be full-length, for example, IgG1, IgG2,IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE antibodies or sequencevariants thereof. Alternatively, the antibodies can be fragments, suchas a Fab, F(ab′)₂, Fv, single chain Fv, isolated complementaritydetermining region (CDR) or a combination of two or more isolated CDRs.The antibodies can be any known type or species of antibody, including,but not limited to, fully human, humanized, and chimeric antibodies.Preferably the antibodies are IgG2 antibodies. It will be appreciatedthat certain modifications may be made to the IgG2 sequence within suchas deletion of the N-terminal lysine and/or various other mutationsknown in the art. Thus an IgG2 antibody includes for example antibodieshaving constant domains with at least 90%, preferably at least 95%,preferably at least 97% and preferably at least 99% sequence identity toa native human IgG2 sequence.

The invention also provides molecular conjugates comprising an antibodyof the invention linked to an antigen (including fragments, epitopes andantigenic determinants), such as a tumor antigen, an autoantigen, or acomponent of a pathogen. For example, the antigen may include a tumorantigen, such as βhCG, gpl00 or Pmel17, CEA, gpl00, TRP-2, NY-BR-1,NY-CO-58, MN (gp250), idiotype, Tyrosinase, Telomerase, SSX2, MUC-1,MAGE-A3, and high molecular weight-melanoma associated antigen (HMW-MAA)MART1, melan-A, NY-ESO-1, MAGE-1, MAGE-3, WT1, Her2, mesothelin or highmolecular weight-melanoma associated antigen (HMW-MAA).

In another embodiment, the molecular complex further includes atherapeutic agent, such as a cytotoxic agent, an immunosuppressiveagent, or a chemotherapeutic agent.

In another aspect, the invention provides bispecific moleculescomprising antibodies of the invention linked to a second functionalmoiety having a different binding specificity. For example, in oneembodiment, the second molecule may bind to a T cell receptor (e.g.,CD3, CD40, or CTLA-4), an NK receptor (e.g., CD56), a B cell receptor(e.g., CD20), or another tumor necrosis factor receptor (e.g., CD95).

Compositions including antibodies, molecular conjugates, or bispecificmolecules described herein, formulated with a pharmaceuticallyacceptable carrier, also are provided. The compositions may furtherinclude an adjuvant, immunostimulatory agent (e.g., CD40 ligand, FLT 3ligand, cytokines, colony-stimulating factors, an anti-CTLA-4 antibody(including without limitation ipilimumab), anti-PD1 antibody (includingwithout limitation MPDL3280A or durvalumab), anti-41BB antibody, antiOX-40 antibody, LPS (endotoxin), ssRNA, dsRNA, Bacille Calmette-Guerin(BCG), Levamisole hydrochloride, intravenous immune globulins and aToll-like Receptor (TLR) agonist (e.g., TLR3 agonist such as Poly IC, aTLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist)), immunosuppressive agent, another antibody, or an antigen,or a STING agonist.

Tumor antigens which can be included in the molecular conjugates orcompositions of the present invention (e.g., in a vaccine, used incombination with an anti-CD40 antibody of the invention) include anyantigen or antigenic determinant which is present on (or associatedwith) a tumor cell and not typically on normal cells, or an antigen orantigenic determinant which is present on or associated with tumor cellsin greater amounts than on normal (non-tumor) cells, or an antigen orantigenic determinant which is present on tumor cells in a differentform than that found on normal (non-tumor) cells. Such antigens includetumor-specific antigens, including tumor-specific membrane antigens,tumor-associated antigens, including tumor-associated membrane antigens,embryonic antigens on tumors, growth factor receptors, growth factorligands, and any other type of antigen that is associated with cancer. Atumor antigen may be, for example, an epithelial cancer antigen, (e.g.,breast, gastrointestinal, lung), a prostate specific cancer antigen(PSA) or prostate specific membrane antigen (PSMA), a bladder cancerantigen, a lung (e.g., small cell lung) cancer antigen, a colon cancerantigen, an ovarian cancer antigen, a brain cancer antigen, a gastriccancer antigen, a renal cell carcinoma antigen, a pancreatic cancerantigen, a liver cancer antigen, an esophageal cancer antigen, a headand neck cancer antigen, or a colorectal cancer antigen. For example,the antigen may include a tumor antigen, such as βhCG, gpl00 or Pmel17,CEA, gpl00, TRP-2, NY-BR-1, NY-CO-58, MN (gp250), idiotype, Tyrosinase,Telomerase, SSX2, MUC-1, MAGE-A3, and high molecular weight-melanomaassociated antigen (HMW-MAA) MART1, melan-A, EGFRvIII, NY-ESO-1, MAGE-1,MAGE-3, WT1, Her2, or mesothelin. Other antigens employed by the presentinvention (e.g., in a vaccine, used in combination with an anti-CD40antibody of the invention) include antigens from infectious diseasepathogens, such as viruses, bacteria, parasites and fungi, examples ofwhich are disclosed herein.

Nucleic acid molecules encoding all or portions of the heavy and/orlight chain variable regions of the antibodies of the invention also areprovided, as well as expression vectors comprising these nucleic acids,and host cells comprising such expression vectors. In one embodiment,the nucleic acid sequences are selected from the group consisting of SEQID NOs: 87-112, respectively, or nucleic acid sequences having e.g., atleast about 85%, 90% or 95% identity to these nucleic acid sequences.

The present invention also provides methods of enhancing an immuneresponse (e.g., a T cell-mediated immune response, and/or an NK-mediatedresponse and/or a B cell-mediated immune response) against an antigen ina subject using the agonistic antibodies described herein. In oneembodiment, the antibodies bind to human CD40 (as expressed on a varietyof immune cell types), thus triggering the cellular proliferation andactivation of antigen-presenting cells (APCs), and activating B-cells,and effector and memory T-cells, which results in enhanced immuneresponses, e.g., against tumor cells. Accordingly, in one embodiment,the methods include administering an antibody (e.g., a full lengthantibody or antigen binding portion thereof), composition or bispecificmolecule of the invention in an amount effective to induce or enhance animmune response against an antigen. In another embodiment, the methodsfurther includes administering the antigen, e.g., simultaneously,separately or sequentially from the antibody, composition, or bispecificmolecule.

Methods for inhibiting the growth of CD40 expressing cells (e.g., in thetreatment of cancers) also are provided. For example, agonisticantibodies of the present invention have been shown to increaseexpression of cell-surface molecules that recruit immune effector cellswhich leads to cell death, e.g., apoptosis. Therefore, in anotherembodiment, the method includes administering or contacting the cellswith the antibody (e.g., a full length antibody or antigen bindingportion thereof), composition or bispecific molecule of the presentinvention in an amount effective to inhibit growth of CD40 expressingcells.

Further provided are methods for targeting an antigen to a cell, e.g., acell capable of antigen presentation (such as peripheral bloodmononuclear cells (PBMC), monocytes (such as THP-1), B lymphoblastoidcells (such as C1R.A2, 1518 B-LCL) and monocyte-derived DCs in a subjectby administering a molecule which binds a receptor on the cell (e.g.,the previously described CD40 antibodies) linked to an antigen.

The methods described herein are useful in treating a variety ofdisorders, particularly cancers (e.g., selected from the groupconsisting of leukemia, acute lymphocytic leukemia, acute myelocyticleukemia, myeloblasts promyelocyte myelomonocytic monocyticerythroleukemia, chronic leukemia, chronic myelocytic(granulocytic)leukemia, chronic lymphocytic leukemia, mantle celllymphoma, primary central nervous system lymphoma, Burkitt's lymphoma,marginal zone B cell lymphoma, Polycythemia vera Lymphoma, Hodgkin'sdisease, non-Hodgkin's disease, multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, solid tumors, sarcomas, andcarcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma,osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, uterine cancer, testicular tumor, lung carcinoma, small celllung carcinoma, non small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basalcell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brainand central nervous system (CNS) cancer, cervical cancer,choriocarcinoma, colorectal cancers, connective tissue cancer, cancer ofthe digestive system, endometrial cancer, esophageal cancer, eye cancer,head and neck cancer, gastric cancer, intraepithelial neoplasm, kidneycancer, larynx cancer, liver cancer, lung cancer (small cell, largecell), melanoma, neuroblastoma; oral cavity cancer (for example lip,tongue, mouth and pharynx), ovarian cancer, pancreatic cancer,retinoblastoma, rhabdomyosarcoma, rectal cancer; cancer of therespiratory system, sarcoma, skin cancer, stomach cancer, testicularcancer, thyroid cancer, uterine cancer, and cancer of the urinarysystem). Particular cancers include CD40-expressing tumors selected fromthe group consisting of chronic lymphocytic leukemia, mantle celllymphoma, primary central nervous system lymphoma, Burkitt's lymphomaand marginal zone B cell lymphoma.

In another embodiment, the methods can be used to treat or prevent abacterial, fungal, viral or parasitic infection

CD40 expressing cells include any and all cells the express CD40,including, but not limited to antigen-presenting cells (APCs), includingdendritic cells (DCs), B-cells, macrophages, and monocytes. CD40 is alsoexpressed on other cell types such as epithelial cells, endothelialcells, and platelets. CD40 expression has been demonstrated on varioustumor cells, including B cell lymphoma and renal cancer cells. In aparticular embodiment, the CD40 expressing cells include cell lines suchas Jurkat cells, Raji cells, Ramos cells and Daudi cells. In anotherembodiment, the CD40 expressing cells are tumor cells or cancer cells.In another embodiment, CD40-expressing cells include B cells, NK cells,T cells that are found infiltrating tumor or cancer cells, also calledtumor infiltrating lymphocytes.

In another embodiment, the invention provides for the use of anantibody, composition or bispecific molecule described herein in themanufacture of a medicament for inducing or enhancing an immune responseagainst an antigen (e.g., a tumor antigen) in a subject. In furtherembodiments, the invention provides for the use of an antibody orcomposition described herein in the manufacture of a medicament for (1)increasing an immune response to an antigen, (2) inhibiting growth ofCD40 expressing cells, and/or (3) targeting an antigen to an APC.

The present invention also provides methods for detecting the presenceor absence of CD40 in a biological sample by (1) contacting a biologicalsample with an antibody described herein (wherein the antibody islabeled with a detectable substance) and (2) detecting the antibodybound to CD40.

Also provided are kits comprising the compositions (e.g., antibodiesand/or bispecific molecules) of the invention and, optionally,instructions for use. The kit can further contain a least one additionalreagent, such as a cytokine or complement, or one or more additionalantibodies of the invention.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the values for the equilibrium dissociation constants(K_(D)) and the kinetic association rate constants (k_(on)) anddissociation rate constants (k_(off)) for antibodies 3C3, 3G5, 1B4, 3B6,and 6H6 as determined by bio-layer interferometry (BLI) using an Octet™QK^(e) instrument (Pall ForteBio, Menlo Park, Calif.) according to themanufacturer's guidelines.

FIG. 2 is a graph showing the binding of human CD40 antibodies(including 3C3, 3G5, 1B4, 3B6, and 6H6) to recombinant purified humanCD40 coated microtiter plates using absorbance (OD₄₅₀) in an ELISA as afunction of antibody concentration.

FIG. 3 are graphs showing the binding as mean fluorescence intensity(MFI) by flow cytometry as a function of human CD40 antibodyconcentration (3C3, 3G5, 1B4, 3B6, and 6H6) to purified human PBMCs(left) and cynomolgus macaque PBMCs (right).

FIGS. 4A and 4B are graphs showing the effect of human CD40 antibodieson the binding of soluble CD40 ligand (sCD40L) to CD40 protein by ELISA.

FIG. 5 is a flow cytometric analysis of human CD40 antibodies (3C3, 3G5,1B4, 3B6, and 6H6) binding to CD40 on Raji cells expressing human CD40on their surface.

FIG. 6 is a flow cytometric analysis of human CD40 antibodies (3C3, 3G5,1B4, 3B6, and 6H6) binding to CD40 on Ramos cells expressing human CD40on their surface.

FIGS. 7A and 7B are graphs showing the induction of CD95 on Ramos cellsby human CD40 antibodies.

FIGS. 8A and 8B are graphs showing dendritic cell (DC) activation byhuman CD40 antibodies (3C3 and 3G5) based on the change in level ofexpression of the following markers: CD54, HLA-DR, CD86, CD83, and %CD83+ cells as indicated.

FIGS. 9A and 9B are graphs showing the induction of IL-12p40 by humanCD40 antibodies (3C3 and 3G5).

FIGS. 10A and 10B are graphs showing B cell activation by human CD40antibodies (3C3 and 3G5) based on the change in level of expression ofthe following markers: CD54, HLA-DR, CD23, % CD23+ cells, CD69, CD86,CD38, and CD71 as indicated.

FIGS. 11A and 11B are graphs depicting NFkB activation by human CD40antibodies using a luciferase reporter cell line expressing CD40.

FIG. 12 are graphs showing the results of tumor growth and survival in aSCID mouse tumor model (Raji cells) following treatment with CD40 humanantibody clones 3C3 and 3G5 via intraperitoneal administration, 0.3 mgper dose.

FIG. 13 are graphs showing the results of tumor growth and survival in aSCID mouse tumor model (Ramos cells) following treatment with CD40 humanantibody clones 3C3 and 3G5 via intraperitoneal administration, 0.3 mgper dose.

FIGS. 14A and 14B are graphs showing T-cell proliferation of labeledPBMCs incubated with CD40 antibodies as indicated or the isotype control(IgG2).

FIG. 15 is a graph showing binding to CD40 independent of Fc receptorinteraction using CD40 antibodies 3C3 and 3G5.

FIG. 16 is a graph showing NFκb activation using CD40 antibodies 3C3 and3G5.

FIG. 17 is a graph showing CD95 induction on Ramos cells using CD40antibodies 3C3 and 3G5.

FIG. 18 shows a schematic representation of an example of ananti-CD40/antigen fusion APC targeted vaccine construct.

FIG. 19 is a graph showing the synergestic effect of CD40 antibody 3C3with soluble CD40L on CD95 expression in Ramon cells.

FIG. 20 is a schematic of soluble CD40 cDNA encoding the full lengthextracellular domain (ECD) spanning amino acid residues 1-173 with anN-terminal human kappa light chain and a C-terminal Flag tag.

FIG. 21 shows an alignment of human CD40 ECD amino acid sequence (SEQ IDNO: 133) with monkey CD40 ECD amino acid sequence (SEQ ID NO: 139)(top)and mouse CD40 ECD amino acid sequence (bottom). Fragments generated areindicated (nucleotides 1-94 of SEQ ID NO: 133, SEQ ID NOS: 140-147).

FIG. 22 provides graphs showing binding of CD40 antibody 3C3 to humanCD40 ECD fragment A (amino acid residues 1-5; top) or fragment D (aminoacid residues 33-36; bottom) with various point mutations orcombinations thereof.

FIGS. 23A-23C are graphs showing levels of separate aminotransferase(AST; 23A), alanine aminotransferase (ALT; 23B) and creatinine kinase(23C) measured in monkeys before and after treatment with CD40antibodies 3C3 or 3G5 at indicated time points.

FIG. 24 is a graph showing levels of IL-12 (pg/mL) measured in bloodfrom monkeys treated with CD40 antibodies 3C3 or 3G5 at indicated timepoints.

FIGS. 25A-25C are graphs showing amounts of white blood cells (25A),neutrophils (25B) and lymphocytes (25C) measured in monkeys before andafter treatment with CD40 antibodies 3C3 or 3G5 at indicated timepoints.

FIG. 26 is a graph showing the percentage change from baseline of amountof B cells in monkeys treated with CD40 antibodies 3C3 or 3G5 over time(days).

FIG. 27 provides graphs showing HLA-DR expression on B cells relative tobaseline following 2 mg (left) or 0.2 mg (right) of CD40 antibody 3C3(square), 3G5 (diamonds) or saline (circles).

FIG. 28 provides a graph showing B-cell proliferation when cells werecultured in the presence of either the anti-CD40 mAb 3C3.

FIGS. 29 and 30 provide graphs showing synergistic effects of thecombination of the anti-CD40 mAb 3C3 and CD40L in B-cells.

FIG. 31 provides a table showing cytokine responses in whole blood whenthis was incubated with the anti-CD40 mAb 3C3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides anti-CD40 antibodies that exhibitparticular functional properties correlating with significanttherapeutic benefits involving upregulation of immune function (e.g. Tcell mediated immune responses as in vaccine therapies, NK activation incancer therapies), inhibition of cell growth (e.g., in cancer therapy),and/or enhanced processing and presentation of an antigen by APCs (e.g.,in vaccine therapy). These functional features include, for example, anincreased immune response to an antigen independent of Fc receptorbinding, and/or without induction of antibody-dependent cellularcytotoxicity (ADCC) or complement dependent cellular cytotoxicity (CDC).Additional functional features include, for example, (1) inhibition of(e.g., complete or partial blocking) binding of CD40L (CD154) to CD40expressing cells by at least 50%, at least 60% or at least 70% (2)blockage of binding of CD40L to human CD40 independent of Fc receptorbinding, (3) induction of cellular apoptosis (e.g., as measured by anincrease in the expression of CD95), (4) increased T-cell stimulatoryactivity (e.g., as measured by an increase in the expression ofIL-12p40), and/or (5) increased B-cell activation (e.g., as measured byan increase in the expression of at least one cell-surface markerselected from the group consisting of HLA-DR V450, CD54 PE, CD86 APC,and CD83 BV510, CD19 V500, CD54 PE, HLA-DR V450, CD23 PerCP-Cy5.5, CD69APC, CD86 APC, CD38 PerCP-Cy5.5 and CD71 PE).

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “CD40” (also referred to as “CD40 molecule,” “Bp50,” “CDW40,”“TNFRSF5,” “p50,” “B cell surface antigen CD40,” “B cell-associatedmolecule,” “CD40 antigen,” “TNF receptor superfamily member 5,” “CD40type II isoform,” “CD40L receptor,” “nerve growth factorreceptor-related B-lymphocyte activation molecule,” or “tumor necrosisfactor receptor superfamily member 5”) refers to a receptor that is amember of the TNF-receptor superfamily, which binds to ligand CD40L(also referred to as CD154). CD40 is mediates a broad variety of immuneand inflammatory responses including T cell-dependent immunoglobulinclass switching, and memory B cell development. The term “CD40” includesany variants or isoforms of CD40 which are naturally expressed by cells(e.g., human CD40 deposited with GENBANK® having accession no. P25942).Accordingly, antibodies of the invention may cross-react with CD40 fromspecies other than human. Alternatively, the antibodies may be specificfor human CD40 and may not exhibit any cross-reactivity with otherspecies. CD40 or any variants and isoforms thereof, may either beisolated from cells or tissues which naturally express them or berecombinantly produced using well-known techniques in the art and/orthose described herein. Preferably the antibodies are targeted to hCD40which has a normal glycosylation pattern. Genbank® (Accession No.P25942) reports the amino acid sequence of human CD40 as follows (SEQ IDNO:1):

MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSLCQPGQKLVSD CTEFTETECL PCGESEFLDT WNRETHCHQHKYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCVLHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEKCHPWTSCETK DLVVQQAGTN KTDVVCGPQD RLRALVVIPIIFGILFAILL VLVFIKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ

The term “CD40L” (also referred to as “CD40 ligand,” “CD407L,” or“CD154”) refers to the ligand for CD40 (see, for example, Schonbeck andLibby (2001) Cell Mol Life Sci, 58(1):4-43). CD40L is primarilyexpressed on activated T cells and is a member of the TNF superfamily ofmolecules. It binds to CD40 on antigen-presenting cells (APC), whichleads to many effects depending on the target cell type (Parham, Peter(2004). The Immune System (2^(nd) ed.). Garland Science. Pp. 169-173).

Genbank® (Accession No. NP_000065) reports the amino acid sequence ofhuman CD40L as follows (SEQ ID NO: 2):

MIETYNQTSP RSAATGLPIS MKIFMYLLTV FLITQMIGSALFAVYLHRRL DKIEDERNLH EDFVFMKTIQ RCNTGERSLSLLNCEEIKSQ FEGFVKDIML NKEETKKENS FEMQKGDQNPQIAAHVISEA SSKTTSVLQW AEKGYYTMSN NLVTLENGKQLTVKRQGLYY IYAQVTFCSN REASSQAPFI ASLCLKSPGRFERILLRAAN THSSAKPCGQ QSIHLGGVFE LQPGASVFVN VTDPSQVSHG TGFTSFGLLK

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers, in one preferred embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., human CD40). Such “fragments” are, for example between about 8and about 1500 amino acids in length, suitably between about 8 and about745 amino acids in length, suitably about 8 to about 300, for exampleabout 8 to about 200 amino acids, or about 10 to about 50 or 100 aminoacids in length. It has been shown that the antigen-binding function ofan antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), CL and CH1 domains;(ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated cmplementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be joined by a synthetic linker. Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies. Antigen-bindingportions can be produced by recombinant DNA techniques, or by enzymaticor chemical cleavage of intact immunoglobulins.

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321(1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term “monoclonal antibody,” as used herein, refers to an antibodywhich displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to an antibody which displays a single binding specificity andwhich has variable and optional constant regions derived from humangermline immunoglobulin sequences. In one embodiment, human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic non-human animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies comprise variable and constant regions that utilizeparticular human germline immunoglobulin sequences are encoded by thegermline genes, but include subsequent rearrangements and mutationswhich occur, for example, during antibody maturation. As known in theart (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), thevariable region contains the antigen binding domain, which is encoded byvarious genes that rearrange to form an antibody specific for a foreignantigen. In addition to rearrangement, the variable region can befurther modified by multiple single amino acid changes (referred to assomatic mutation or hypermutation) to increase the affinity of theantibody to the foreign antigen. The constant region will change infurther response to an antigen (i.e., isotype switch). Therefore, therearranged and somatically mutated nucleic acid molecules that encodethe light chain and heavy chain immunoglobulin polypeptides in responseto an antigen may not have sequence identity with the original nucleicacid molecules, but instead will be substantially identical or similar(i.e., have at least 80% identity).

The term “human antibody” includes antibodies having variable andconstant regions (if present) of human germline immunoglobulinsequences. Human antibodies of the invention can include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo) (see, Lonberg, N. et al. (1994) Nature368(6474): 856-859); Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann.N.Y. Acad. Sci 764:536-546). However, the term “human antibody” does notinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences (i.e., humanized antibodies).

As used herein, a “heterologous antibody” is defined in relation to thetransgenic non-human organism producing such an antibody. This termrefers to an antibody having an amino acid sequence or an encodingnucleic acid sequence corresponding to that found in an organism notconsisting of the transgenic non-human animal, and generally from aspecies other than that of the transgenic non-human animal.

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to human CD40 is substantially free of antibodiesthat specifically bind antigens other than human CD40). An isolatedantibody that specifically binds to an epitope of may, however, havecross-reactivity to other CD40 proteins from different species. However,the antibody preferably always binds to human CD40. In addition, anisolated antibody is typically substantially free of other cellularmaterial and/or chemicals. In one embodiment of the invention, acombination of “isolated” antibodies having different CD40 specificitiesis combined in a well defined composition.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents, whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 amino acids in a unique spatial conformation. Methods for determiningwhat epitopes are bound by a given antibody (i.e., epitope mapping) arewell known in the art and include, for example, immunoblotting andimmunoprecipitation assays, wherein overlapping or contiguous peptidesfrom CD40 are tested for reactivity with the given anti-CD40 antibody.Methods of determining spatial conformation of epitopes includetechniques in the art and those described herein, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance (see, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

Accordingly, antibodies that bind to the same epitope, or an epitope onCD40 which comprises all or a portion of an epitope recognized by theparticular antibodies described herein (e.g., the same or an overlappingregion or a region between or spanning the region) also are provided bythe invention. Antibodies that bind to the same epitope, or an epitopewhich comprises all or a portion of an epitope recognized by particularantibody can be identified using routine techniques. Such techniquesinclude, for example, epitope mapping methods, such as, x-ray analysesof crystals of antigen:antibody complexes which provides atomicresolution of the epitope. Other methods monitor the binding of theantibody to antigen fragments or mutated variations of the antigen whereloss of binding due to a modification of an amino acid residue withinthe antigen sequence is often considered an indication of an epitopecomponent. In addition, computational combinatorial methods for epitopemapping can also be used. These methods rely on the ability of theantibody of interest to affinity isolate specific short peptides fromcombinatorial phage display peptide libraries. The peptides are thenregarded as leads for the definition of the epitope corresponding to theantibody used to screen the peptide library. For epitope mapping,computational algorithms have also been developed which have been shownto map conformational discontinuous epitopes.

Also provided are antibodies that compete for binding to human CD40 withthe antibodies described herein. Antibodies that compete for binding canbe identified using routine techniques. Such techniques include, forexample, an immunoassay, which shows the ability of one antibody toblock the binding of another antibody to a target antigen, i.e., acompetitive binding assay. Competitive binding is determined in an assayin which the immunoglobulin under test inhibits specific binding of areference antibody to a common antigen, such as CD40. Numerous types ofcompetitive binding assays are known, for example: solid phase direct orindirect radioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using I-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore.

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen. Typically, the antibody bindswith an equilibrium dissociation constant (K_(D)) of approximately lessthan 10⁻⁷ M, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ Mor even lower when determined by bio-layer interferometry (BLI) using anOctet™ QK^(e) instrument or by surface plasmon resonance (SPR)technology in a BIACORE 2000 instrument using recombinant human CD40 asthe analyte and the antibody as the ligand and binds to thepredetermined antigen with an affinity that is at least two-fold greaterthan its affinity for binding to a non-specific antigen (e.g., BSA,casein) other than the predetermined antigen or a closely-relatedantigen. The phrases “an antibody recognizing an antigen” and “anantibody specific for an antigen” are used interchangeably herein withthe term “an antibody which binds specifically to an antigen.”

Also, encompassed by the present invention are antibodies that bind tohuman CD40 and are capable of increasing an immune response independentof Fc receptor binding. For example, such antibodies exhibit potentagonistic features without cross-linking with an Fc receptor, such asFcγR. These agonistic features include, for example, an increase inT-cell activity and/or an increase in B cell activation as measured,e.g., by an increase in the expression of cell surface markers.

The term “K_(D),” as used herein, is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction. Typically, the human antibodies of the invention bind toCD40 with a dissociation equilibrium constant (K_(D)) of approximately10⁻⁸ M or less, such as less than 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M,or even lower when determined by bio-layer interferometry (BLI) using anOctet™ QK^(e) instrument or by surface plasmon resonance (SPR)technology in a BIACORE 2000 instrument using recombinant human CD40 asthe analyte and the antibody as the ligand.

The term “kd” as used herein, is intended to refer to the off rateconstant for the dissociation of an antibody from the antibody/antigencomplex.

The term “ka” as used herein, is intended to refer to the on rateconstant for the association of an antibody with the antigen.

The term “EC50,” as used herein, refers to the concentration of anantibody or an antigen-binding portion thereof, which induces aresponse, either in an in vitro or an in vivo assay, which is 50% of themaximal response, i.e., halfway between the maximal response and thebaseline.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes. In oneembodiment, a human monoclonal antibody of the invention is of the IgG1isotype. In another embodiment, a human monoclonal antibody of theinvention is of the IgG2 isotype.

The term “binds to immobilized CD40,” refers to the ability of a humanantibody of the invention to bind to CD40, for example, expressed on thesurface of a cell or which is attached to a solid support.

The term “cross-reacts,” as used herein, refers to the ability of anantibody of the invention to bind to CD40 from a different species. Forexample, an antibody of the present invention which binds human CD40 mayalso bind another species of CD40. As used herein, cross-reactivity ismeasured by detecting a specific reactivity with purified antigen inbinding assays (e.g., SPR, ELISA) or binding to, or otherwisefunctionally interacting with, cells physiologically expressing CD40.Methods for determining cross-reactivity include standard binding assaysas described herein, for example, by bio-layer interferometry (BLI)using an Octet™ QK^(e) instrument or by Biacore™ surface plasmonresonance (SPR) analysis using a Biacore™ 2000 SPR instrument (BiacoreAB, Uppsala, Sweden), or flow cytometric techniques.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

As used herein, “nonswitched isotype” refers to the isotypic class ofheavy chain that is produced when no isotype switching has taken place;the CH gene encoding the nonswitched isotype is typically the first CHgene immediately downstream from the functionally rearranged VDJ gene.Isotype switching has been classified as classical or non-classicalisotype switching. Classical isotype switching occurs by recombinationevents which involve at least one switch sequence region in thetransgene. Non-classical isotype switching may occur by, for example,homologous recombination between human a and human Σ_(μ) (δ-associateddeletion). Alternative non-classical switching mechanisms, such asintertransgene and/or interchromosomal recombination, among others, mayoccur and effectuate isotype switching.

As used herein, the term “switch sequence” refers to those DNA sequencesresponsible for switch recombination. A “switch donor” sequence,typically a μ switch region, will be 5′ (i.e., upstream) of theconstruct region to be deleted during the switch recombination. The“switch acceptor” region will be between the construct region to bedeleted and the replacement constant region (e.g., γ, ε, etc.). As thereis no specific site where recombination always occurs, the final genesequence will typically not be predictable from the construct.

As used herein, “glycosylation pattern” is defined as the pattern ofcarbohydrate units that are covalently attached to a protein, morespecifically to an immunoglobulin protein. A glycosylation pattern of aheterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the nonhuman transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the nonhuman transgenic animal than tothe species from which the CH genes of the transgene were derived.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete V_(H) or V_(L) domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., V_(H),V_(L), CDR3) that bind to CD40, is intended to refer to a nucleic acidmolecule in which the nucleotide sequences encoding the antibody orantibody portion are free of other nucleotide sequences encodingantibodies or antibody portions that bind antigens other than CD40,which other sequences may naturally flank the nucleic acid in humangenomic DNA.

The present invention also encompasses “conservative sequencemodifications” of the sequences set forth in SEQ ID Nos: 3-132, i.e.,nucleotide and amino acid sequence modifications which do not abrogatethe binding of the antibody encoded by the nucleotide sequence orcontaining the amino acid sequence, to the antigen. Such conservativesequence modifications include conservative nucleotide and amino acidsubstitutions, as well as, nucleotide and amino acid additions anddeletions. For example, modifications can be introduced into SEQ ID Nos:3-148 by standard techniques known in the art, such as site-directedmutagenesis and PCR-mediated mutagenesis. Conservative amino acidsubstitutions include ones in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in a human anti-CD40 antibody is preferably replacedwith another amino acid residue from the same side chain family. Methodsof identifying nucleotide and amino acid conservative substitutionswhich do not eliminate antigen binding are well-known in the art (see,e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al.Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad.Sci. USA 94:412-417 (1997)).

Conservative substitutions maybe made, for example, according to theTable below. For example, amino acids in the same block in the secondcolumn and preferably in the same line in the third column may besubstituted for each other.

Aliphatic Non-Polar GAP ILV Polar-uncharged CSTM NQ Polar-charged DE KRAromatic HFWY

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of an anti-CD40 antibody coding sequence,such as by saturation mutagenesis, and the resulting modified anti-CD40antibodies can be screened for binding activity.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available atwww.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50,60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percentidentity between two nucleotide or amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997)Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures thereof may be mutated, inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”) In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

As used herein, the term “antigen” refers to any natural or syntheticimmunogenic substance, such as a protein, peptide, or hapten. Suitableantigens for use in the present invention (e.g., in a vaccine incombination with an anti-CD40 antibody of the invention) include, forexample, infectious disease antigens and tumor antigens, against whichprotective or therapeutic immune responses are desired, e.g., antigensexpressed by a tumor cell or a pathogenic organism or infectious diseaseantigens. For example, suitable antigens include tumor-associatedantigens for the prevention or treatment of cancers. Examples oftumor-associated antigens include, but are not limited to, sequencescomprising all or part of the sequences of βhCG, gpl00 or Pmel17,HER2/neu, WT1, mesothelin, CEA, gpl00, MART1, TRP-2, melan-A, NY-ESO-1,NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MAGE-3, MAGE-A3,Tyrosinase, Telomerase, SSX2 and MIUC-1 antigens, and germ cell derivedtumor antigens. Tumor associated antigens also include the blood groupantigens, for example, Le^(a), Le^(b), LeX, LeY, H-2, B-1, B-2 antigens.Alternatively, more than one antigen can be included within theantigen-antibody constructs of the invention. For example, a MAGEantigen can be combined with other antigens such as melanin A,tyrosinase, and gpl00 along with adjuvants such as GM-CSF or IL-12, andlinked to an anti-APC antibody.

Other suitable antigens include viral antigens for the prevention ortreatment of viral diseases. Examples of viral antigens include, but arenot limited to, HIV-1 gag, HIV-1 env, HIV-1 nef, HBV (surface or coreantigens), HPV, FAS, HSV-1, HSV-2, p17, ORF2 and ORF3 antigens. Examplesof bacterial antigens include, but are not limited to, Toxoplasma gondiior Treponema pallidum. The antibody-bacterial antigen conjugates of theinvention can be in the treatment or prevention of various bacterialdiseases such as Anthrax, Botulism, Tetanus, Chlamydia, Cholera,Diphtheria, Lyme Disease, Syphilis and Tuberculosis. Other suitableantigens from infectious disease pathogens, such as viruses, bacteria,parasites and fungi are disclosed below.

Sequences of the foregoing antigens are well known in the art. Forexample, an example of a MAGE-3 cDNA sequence is provided in U.S. Pat.No. 6,235,525 (Ludwig Institute for Cancer Research); examples ofNY-ESO-1 nucleic acid and protein sequences are provided in U.S. Pat.Nos. 5,804,381 and 6,069,233 (Ludwig Institute for Cancer Research);examples of Melan-A nucleic acid and protein sequences are provided inU.S. Pat. Nos. 5,620,886 and 5,854,203 (Ludwig Institute for CancerResearch); examples of NY-BR-1 nucleic acid and protein sequences areprovided in U.S. Pat. Nos. 6,774,226 and 6,911,529 (Ludwig Institute forCancer Research) and examples of NY-CO-58 nucleic acid and proteinsequences are provided in WO 02090986 (Ludwig Institute for CancerResearch); an example of an amino acid sequence for the HER-2/neuprotein is available at GENBANK® Accession No. AAA58637; and anucleotide sequence (mRNA) for human carcinoembryonic antigen-like 1(CEA-1) is available at GENBANK® Accession No. NM_020219.

An HPV antigen that may be used in the compositions and the methods ofthe invention may include, for example an HPV-16 antigen, an HPV-18antigen, an HPV-31 antigen, an HPV-33 antigen and/or an HPV-35 antigen;and is suitably an HPV-16 antigen and/or HPV-18 antigen. A genome ofHPV-16 is described in Virology, 145:181-185 (1985) and DNA sequencesencoding HPV-18 are described in U.S. Pat. No. 5,840,306, thedisclosures of which are incorporated by reference herein in theirentirety. HPV-16 antigens (e.g., seroreactive regions of the E1 and/orE2 proteins of HPV-16) are described in U.S. Pat. No. 6,531,127, andHPV-18 antigens (e.g., seroreactive regions of the L1 and/or L2 proteinsof HPV-18) are described in U.S. Pat. No. 5,840,306, the disclosures ofwhich are incorporated by reference herein. Similarly, a complete genomefor HBV is available at GENBANK® Accession No. NC_003977, the disclosureof which is incorporated herein. The genome of HCV is described inEuropean Patent Application No. 318 216, the disclosure of which isincorporated herein. PCT/US90/01348, incorporated by reference herein,discloses sequence information of clones of the HCV genome, amino acidsequences of HCV viral proteins and methods of making and using suchcompositions for HCV vaccines comprising HCV proteins and peptidesderived there from.

Antigenic peptides of proteins (i.e., those containing T cell epitopes)can be identified in a variety of manners well known in the art. Forexample, T cell epitopes can be predicted by analyzing the sequence ofthe protein using web-based predictive algorithms (BIMAS & SYFPEITHI) togenerate potential MHC class I and II-binding peptides that match aninternal database of 10,000 well characterized MHC binding peptidespreviously defined by CTLs. High scoring peptides can be ranked andselected as “interesting” on the basis of high affinity to a given MHCmolecule.

Another method for identifying antigenic peptides containing T cellepitopes is by dividing the protein into non-overlapping peptides ofdesired length or overlapping peptides of desired lengths which can beproduced recombinantly, synthetically, or in certain limited situations,by chemical cleavage of the protein and tested for immunogenicproperties, e.g., eliciting a T cell response (i.e., proliferation orlymphokine secretion).

In order to determine precise T cell epitopes of the protein by, forexample, fine mapping techniques, a peptide having T cell stimulatingactivity and thus comprising at least one T cell epitope, as determinedby T cell biology techniques, can be modified by addition or deletion ofamino acid residues at either the amino or carboxy terminus of thepeptide and tested to determine a change in T cell reactivity to themodified peptide. If two or more peptides which share an area of overlapin the native protein sequence are found to have human T cellstimulating activity, as determined by T cell biology techniques,additional peptides can be produced comprising all or a portion of suchpeptides and these additional peptides can be tested by a similarprocedure. Following this technique, peptides are selected and producedrecombinantly or synthetically. Peptides are selected based on variousfactors, including the strength of the T cell response to the peptide(e.g., stimulation index). The physical and chemical properties of theseselected peptides (e.g., solubility, stability) can then be examined todetermine whether the peptides are suitable for use in therapeuticcompositions or whether the peptides require modification.

The term “antigen presenting cell” or “APC” is a cell that displaysforeign antigen complexed with MHC on its surface. T-cells recognizethis complex using T-cell receptor (TCR). Examples of APCs include, butare not limited to, dendritic cells (DCs), peripheral blood mononuclearcells (PBMC), monocytes (such as THP-1), B lymphoblastoid cells (such asC1R.A2, 1518 B-LCL) and monocyte-derived dendritic cells (DCs). SomeAPCs internalize antigens either by phagocytosis or by receptor-mediatedendocytosis. Examples of APC receptors include, but are not limited toC-type lectins, such as, the human Dendritic and Epithelial Cell 205receptor (DEC-205), and the human macrophage mannose receptor.

The term “antigen presentation” refers to the process by which APCscapture antigens and enables their recognition by T-cells, e.g., as acomponent of an MHC-I and/or MHC-II conjugate.

“MHC molecules” include two types of molecules, MHC class I and MHCclass II. MHC class I molecules present antigen to specific CD8+ T cellsand MHC class II molecules present antigen to specific CD4+ T cells.Antigens delivered exogenously to APCs are processed primarily forassociation with MHC class II. In contrast, antigens deliveredendogenously to APCs are processed primarily for association with MHCclass I.

As used herein, the term “immunostimulatory agent” includes but is notlimited to compounds capable of stimulating APCs, such as DCs andmacrophages. For example, suitable immunostimulatory agents for use inthe present invention are capable of stimulating APCs, so that thematuration process of the APCs is accelerated, the proliferation of APCsis increased, and/or the recruitment or release of co-stimulatorymolecules (e.g., CD80, CD86, ICAM-1, MHC molecules and CCR7) andpro-inflammatory cytokines (e.g., IL-10, IL-6, IL-12, IL-15, and IFN-γ)is upregulated. Suitable immunostimulatory agents are also capable ofincreasing T cell proliferation. Such immunostimulatory agents include,but are not be limited to, CD27 ligand; FLT 3 ligand; cytokines, such asIFN-α, IFN-β, IFN-γ and IL-2; colony-stimulating factors, such as G-CSF(granulocyte colony-stimulating factor) and GM-CSF(granulocyte-macrophage colony-stimulating factor); an anti-CTLA-4antibody, anti-PD1 antibody, anti-41BB antibody, or anti-OX-40 antibody;LPS (endotoxin); ssRNA; dsRNA; Bacille Calmette-Guerin (BCG); Levamisolehydrochloride; and intravenous immune globulins. In one embodiment animmunostimulatory agent may be a Toll-like Receptor (TLR) agonist. Forexample the immunostimulatory agent may be a TLR3 agonist such asdouble-stranded inosine:cytosine polynucleotide (Poly I:C, for exampleavailable as Ampligen™ from Hemispherx Bipharma, PA, US or Poly IC:LCfrom Oncovir) or Poly A:U; a TLR4 agonist such as monophosphoryl lipid A(MPL) or RC-529 (for example as available from GSK, UK); a TLR5 agonistsuch as flagellin; a TLR7 or TLR8 agonist such as an imidazoquinolineTLR7 or TLR 8 agonist, for example imiquimod (e.g., Aldara™) orresiquimod and related imidazoquinoline agents (for example as availablefrom 3M Corporation); or a TLR 9 agonist such as a deoxynucleotide withunmethylated CpG motifs (so-called “CpGs”, for example as available fromColey Pharmaceutical). A preferred immunostimulatory agent is a TLR3agonist, preferably Poly I:C. Such immunostimulatory agents may beadministered simultaneously, separately or sequentially with theantibodies and constructs of the present invention and may also bephysically linked to the antibodies and constructs.

As used herein, the term “linked” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagealso can be genetic (i.e., recombinantly fused). Such linkages can beachieved using a wide variety of art recognized techniques, such aschemical conjugation and recombinant protein production.

As used herein, the term antigen “cross-presentation” refers topresentation of exogenous protein antigens to T cells via MHC class Iand class II molecules on APCs.

As used herein, the term “T cell-mediated response” refers to anyresponse mediated by T cells, including effector T cells (e.g., CD8⁺cells) and helper T cells (e.g., CD4⁺ cells). T cell mediated responsesinclude, for example, T cell cytotoxicity and proliferation.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8⁺ T cells.

As used herein, the terms “inhibits” or “blocks” (e.g., referring toinhibition/blocking of binding of CD40L to CD40 on cells) are usedinterchangeably and encompass both partial and completeinhibition/blocking. The inhibition/blocking of CD40L preferably reducesor alters the normal level or type of activity that occurs when CD40Lbinding occurs without inhibition or blocking. Inhibition and blockingare also intended to include any measurable decrease in the bindingaffinity of CD40L when in contact with an anti-CD40 antibody as comparedto CD40L not in contact with an anti-CD40 antibody, e.g., inhibitsbinding of CD40L by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100%. In a particular embodiment, the anti-CD40 antibodyinhibits binding of CD40L by at least about 70% as measured, e.g., by aBLI or SPR (Biacore) assay. In another embodiment, the anti-CD40antibody inhibits binding of CD40L by at least about 80%.

As used herein, the term “inhibits growth” (e.g., referring to cells) isintended to include any measurable decrease in the growth of a cell,e.g., the inhibition of growth of a cell by at least about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

The terms “inducing an immune response,” “increasing an immuneresponse,” and “enhancing an immune response” are used interchangeablyand refer the stimulation of an immune response (i.e., either passive oradaptive) to a particular antigen.

The terms “induce” and “increase” as used with respect to inducing CDCor ADCC refer to the stimulation of particular direct cell killingmechanisms. For example, in one embodiment, the antibody induces atleast about 20, 25, 30, 35, 40, 45, 50, 55, or 60% lysis via CDC of CD40expressing cells at a concentration of 10 μg/ml. In a preferredembodiment, the antibody induces at least about 40% lysis via CDC ofCD40 expressing cells at a concentration of 10 g/ml. In anotherembodiment, the antibody induces at least about 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, or 85% lysis via ADCC (i.e., specific lysis)of CD40 expressing cells at a concentration of 10 μg/ml. In oneembodiment, the antibody induces at least about 40% lysis via ADCC ofCD40 expressing cells at a concentration of 10 μg/ml.

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, in need of suchtreatment, a human antibody of the present invention, for example, asubject in need of an enhanced immune response against a particularantigen or a subject who ultimately may acquire such a disorder, inorder to prevent, cure, delay, reduce the severity of, or ameliorate oneor more symptoms of the disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the disorderbeing treated and the general state of the patient's own immune system.

As used herein, the term “synergistic” means that administration of twodrugs produce a greater effect when used in combination than would beexpected from adding the individual effects of the two components, forexample greater than two times, greater than three times, greater thanfive times or greater than ten times what would be expected from addingthe individual effects of the two components. For example, druginteractions can be analyzed using the commercial software packageCalcusyn, which is based on the median effect model of Chou and Talalay(Chou, T. C. & Talalay, P. (1984) Adv. Enzyme Regul. 22, 27-55.Quantatative analysis of dose-effect relationships: the combined effectsof multiple drugs or enzyme inhibitors). A Combination Index (C.I.) of 1indicated an additive drug interaction, whereas a C.I. greater than 1was antagonistic and a score lower than 1 was synergistic. The CI valuedefinitions are as follows: 1.45-1.2 is moderately antagonistic, 1.2-1.1is slightly antagonistic, 1.1-0.9 is additive, 0.9-0.85 is slightlysynergistic, 0.85-0.7 is moderately synergistic and 0.7-0.3 issynergistic.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions of the presentinvention can be used to treat a subject with an immune disorder. Theterm “non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dog, cow, chickens,amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Production of Antibodies to CD40

Anti-CD40 antibodies of the invention can be produced using a variety ofknown techniques, such as the standard somatic cell hybridizationtechnique described by Kohler and Milstein, Nature 256: 495 (1975).Although somatic cell hybridization procedures can be used, inprinciple, other techniques for producing monoclonal antibodies also canbe employed, e.g., viral or oncogenic transformation of B lymphocytes,phage display technique using libraries of human antibody genes.

In a particular (exemplified) embodiment, a mouse (e.g., an H2L2 strainof Harbour® transgenic mice) or other appropriate host animal isimmunized with a suitable antigen in order to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the antigen used for immunization. Alternatively, lymphocytesmay be immunized in vitro. Lymphocytes can then be fused with myelomacells using a suitable fusing agent, such as polyethylene glycol, toform a hybridoma cell (Goding, Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986)). Culture medium in whichhybridoma cells are growing is assayed for production of monoclonalantibodies directed against the antigen. After hybridoma cells areidentified that produce antibodies of the desired specificity, affinity,and/or activity, the clones may be subcloned by limiting dilutionprocedures and grown by standard methods (Goding, MonoclonalAntibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986)).Suitable culture media for this purpose include, for example, D-MEM orRPMI-1640 medium. In addition, the hybridoma cells may be grown in vivoas ascites tumors in an animal. The monoclonal antibodies secreted bythe subclones can be separated from the culture medium, ascites fluid,or serum by conventional immunoglobulin purification procedures such as,for example, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

In another embodiment, antibodies directed against CD40 are generatedusing transgenic or transchromosomal mice carrying parts of the humanimmune system rather than the mouse system. In one embodiment, theinvention employs transgenic mice, referred to herein as “HuMAb mice”which contain a human immunoglobulin gene miniloci that encodesunrearranged human heavy (μ and γ) and κ light chain immunoglobulinsequences, together with targeted mutations that inactivate theendogenous μ and κ chain loci (Lonberg, N. et al. (1994) Nature368(6474): 856-859). Accordingly, the mice exhibit reduced expression ofmouse IgM or κ, and in response to immunization, the introduced humanheavy and light chain transgenes undergo class switching and somaticmutation to generate high affinity human IgGκ monoclonal antibodies(Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994)Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F.and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546). The preparationof HuMAb mice is described in detail in Section II below and in Taylor,L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al.(1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc.Nat. Acad. Sci USA 90:3720-3724; Choi et al. (1993) Nature Genetics4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al.(1994) J Immunol. 152:2912-2920; Lonberg et al., (1994) Nature368(6474): 856-859; Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Taylor, L. et al. (1994) InternationalImmunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N.Y.Acad. Sci 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology14: 845-851. See further, U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;5,874,299; and 5,770,429; all to Lonberg and Kay, and GenPharmInternational; U.S. Pat. No. 5,545,807 to Surani et al.; InternationalPublication Nos. WO 98/24884, published on Jun. 11, 1998; WO 94/25585,published Nov. 10, 1994; WO 93/1227, published Jun. 24, 1993; WO92/22645, published Dec. 23, 1992; WO 92/03918, published Mar. 19, 1992.

In another embodiment, antibodies that bind human CD40 can be isolatedfrom antibody phage libraries generated using the techniques describedin, for example, McCafferty et al., Nature, 348:552-554 (1990). Clacksonet al., Nature, 352:624-628 (1991), Marks et al., J. Mol. Biol.,222:581-597 (1991) and Hoet et al (2005) Nature Biotechnology 23,344-348; U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner etal.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat.Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos.5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 toGriffiths et al. Additionally, production of high affinity (nM range)human antibodies by chain shuffling (Marks et al., Bio/Technology,10:779-783 (1992)), as well as combinatorial infection and in vivorecombination as a strategy for constructing very large phage libraries(Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)) may also beused.

In a particular embodiment, the antibody that binds human CD40 isproduced using the phage display technique described by Hoet et al.,supra. This technique involves the generation of a human Fab libraryhaving a unique combination of immunoglobulin sequences isolated fromhuman donors and having synthetic diversity in the heavy-chain CDRs isgenerated. The library is then screened for Fabs that bind to humanCD40.

The preferred animal system for generating hybridomas which produceantibodies of the invention is the murine system. Hybridoma productionin the mouse is well known in the art, including immunization protocolsand techniques for isolating and fusing immunized splenocytes.

Generation of Transfectomas Producing Monoclonal Antibodies to CD40

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(Morrison, S. (1985) Science 229:1202).

For example, in one embodiment, the gene(s) of interest, e.g., humanantibody genes, can be ligated into an expression vector such as aeukaryotic expression plasmid such as used by GS gene expression systemdisclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expressionsystems well known in the art. The purified plasmid with the clonedantibody genes can be introduced in eukaryotic host cells such asCHO-cells or NSO-cells or alternatively other eukaryotic cells like aplant derived cells, fungi or yeast cells. The method used to introducethese genes could be methods described in the art such aselectroporation, lipofectine, lipofectamine or other. After introducingthese antibody genes in the host cells, cells expressing the antibodycan be identified and selected. These cells represent the transfectomaswhich can then be amplified for their expression level and upscaled toproduce antibodies. Recombinant antibodies can be isolated and purifiedfrom these culture supernatants and/or cells.

Alternatively these cloned antibody genes can be expressed in otherexpression systems such as E. coli or in complete organisms or can besynthetically expressed.

Use of Partial Antibody Sequences to Express Intact Antibodies

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998, Nature332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C.et al., 1989, Proc. Nat. Acad. See. U.S.A. 86:10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V(D)J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual evenly across the variable region. For example, somaticmutations are relatively infrequent in the amino-terminal portion offramework region. For example, somatic mutations are relativelyinfrequent in the amino terminal portion of framework region 1 and inthe carboxy-terminal portion of framework region 4. Furthermore, manysomatic mutations do not significantly alter the binding properties ofthe antibody. For this reason, it is not necessary to obtain the entireDNA sequence of a particular antibody in order to recreate an intactrecombinant antibody having binding properties similar to those of theoriginal antibody (see PCT/US99/05535 filed on Mar. 12, 1999). Partialheavy and light chain sequence spanning the CDR regions is typicallysufficient for this purpose. The partial sequence is used to determinewhich germline variable and joining gene segments contributed to therecombined antibody variable genes. The germline sequence is then usedto fill in missing portions of the variable regions. Heavy and lightchain leader sequences are cleaved during protein maturation and do notcontribute to the properties of the final antibody. To add missingsequences, cloned cDNA sequences can be combined with syntheticoligonucleotides by ligation or PCR amplification. Alternatively, theentire variable region can be synthesized as a set of short,overlapping, oligonucleotides and combined by PCR amplification tocreate an entirely synthetic variable region clone. This process hascertain advantages such as elimination or inclusion or particularrestriction sites, or optimization of particular codons.

The nucleotide sequences of heavy and light chain transcripts from ahybridoma are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266:19867-19870); and, HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding, and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotide approximately the midpoint of the correspondingnon-coding oligonucleotide. Thus, for each chain, the oligonucleotidescan be assembled into overlapping double stranded sets that spansegments of 150-400 nucleotides. The pools are then used as templates toproduce PCR amplification products of 150-400 nucleotides. Typically, asingle variable region oligonucleotide set will be broken down into twopools which are separately amplified to generate two overlapping PCRproducts. These overlapping products are then combined by PCRamplification to form the complete variable region. It may also bedesirable to include an overlapping fragment of the heavy or light chainconstant region (including the BbsI site of the kappa light chain, orthe AgeI site if the gamma heavy chain) in the PCR amplification togenerate fragments that can easily be cloned into the expression vectorconstructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, leader sequence, translation initiation,leader sequence, constant region, 3′ untranslated, polyadenylation, andtranscription termination, sequences to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form ahost cell expressing both chains.

Plasmids for use in construction of expression vectors were constructedso that PCR amplified V heavy and V kappa light chain cDNA sequencescould be used to reconstruct complete heavy and light chain minigenes.These plasmids can be used to express completely human IgG₁κ or IgG₄κantibodies. Fully human and chimeric antibodies of the present inventionalso include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similarplasmids can be constructed for expression of other heavy chainisotypes, or for expression of antibodies comprising lambda lightchains.

Thus, in another aspect of the invention, structural features ofanti-CD40 antibodies of the invention are used to create structurallyrelated anti-CD40 antibodies that retain at least one functionalproperty of the antibodies of the invention, such as, for example,

(a) inducing or enhancing an immune response to an antigen independentof Fc receptor binding;

(b) inducing or enhancing an immune response to an antigen withoutinducing antibody-dependent cellular cytotoxicity (ADCC) of CD40expressing cells;

(c) inducing or enhancingan immune response to an antigen withoutinducing complement dependent cellular cytotoxicity (CDC) of CD40expressing cells; and/or

(d) capable of synergising with CD40L; and/or Additional features mayinclude, for example:

(d) no inhibiting or no blocking binding of CD40L;

(e) inhibiting or blocking binding of CD40L;

(f) inhibiting or blocking binding of CD40L to human CD40 independent ofFc receptor binding;

(g) inducing or enhancing cellular apoptosis of a tumor cell;

(h) inducing or enhancing T-cell stimulatory activity of a cell (e.g.,as measured by an increase in the expression of IL-12p40); and/or

(i) inducing or enhancing B-cell activation (e.g., as measured by anincrease in the expression of at least one cell-surface marker selectedfrom the group consisting of HLA-DR V450, CD54 PE, CD86 APC, and CD83BV510, CD19 V500, CD54 PE, HLA-DR V450, CD23 PerCP-Cy5.5, CD69 APC, CD86APC, CD38 PerCP-Cy5.5 and CD71 PE).

In one embodiment, one or more CDR regions of antibodies of theinvention can be combined recombinantly with known framework regions andCDRs to create additional, recombinantly-engineered, anti-CD40antibodies of the invention. The heavy and light chain variableframework regions can be derived from the same or different antibodysequences. The antibody sequences can be the sequences of naturallyoccurring antibodies or can be consensus sequences of severalantibodies. See Kettleborough et al., Protein Engineering 4:773 (1991);Kolbinger et al., Protein Engineering 6:971 (1993) and Carter et al., WO92/22653.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-CD40 antibody including: preparing an antibodyincluding (1) heavy chain framework regions and heavy chain CDRs, whereat least one of the heavy chain CDRs includes an amino acid sequenceselected from the amino acid sequences of CDRs shown in SEQ ID NOs:5, 6,7, 8, 9, 10, 19, 20, 21, 22, 23, 24, 33, 34, 35, 36, 37, 38, 47, 48, 49,51, 52, 61, 62, 63, 64, 65, 66, 75, 76, 77, 78, 79, 80, 89, 90, 91, 92,93, 94, 103, 104, 105, 106, 107, 108; and (2) light chain frameworkregions and light chain CDRs, where at least one of the light chain CDRsincludes an amino acid sequence selected from the amino acid sequencesof CDRs shown in SEQ ID NOs:11, 12, 13, 14, 15, 16, 25, 26, 27, 28, 29,30, 39, 40, 41, 42, 43, 44, 53, 54, 55, 56, 57, 58, 67, 68, 69, 70, 71,72, 81, 82, 83, 84, 85, 86, 95, 96, 97, 98, 99, 100, 109, 110, 111, 112,113, 114; where the antibody retains the ability to bind to CD40. Theability of the antibody to bind CD40 can be determined using standardbinding assays, such as those set forth in the Examples (e.g., an ELISAor a FLISA).

It is well known in the art that antibody heavy and light chain CDR3domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen (see, Hall et al., JImunol., 149:1605-1612 (1992); Polymenis et al., J Immunol.,152:5318-5329 (1994); Jahn et al., Immunobiol., 193:400-419 (1995);Klimka et al., Brit. J. Cancer, 83:252-260 (2000); Beiboer et al., JMol. Biol, 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA,95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc., 116:2161-2162(1994); Ditzel et al., J. Immunol., 157:739-749 (1996)). Accordingly,the recombinant antibodies of the invention prepared as set forth abovepreferably comprise the heavy and/or light chain CDR3s of antibodies3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, and 3B6-NS. The antibodiesfurther can comprise the CDR2s of antibodies 3C3, 3G5, 1B4, 3B6, 6H6,2E1.2, 1B5-NK, and 3B6-NS. The antibodies further can comprise the CDR1sof antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, and 3B6-NS. Theantibodies can further comprise any combinations of the CDRs.

Accordingly, in another embodiment, the invention further providesanti-CD40 antibodies comprising: (1) heavy chain framework regions, aheavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chainCDR3 region, wherein the heavy chain CDR3 region is selected from theCDR3s of 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, or 3B6-NS, and (2)light chain framework regions, a light chain CDR1 region, a light chainCDR2 region, and a light chain CDR3 region, wherein the light chain CDR3region is selected from the CDR3s of 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2,1B5-NK, or 3B6-NS, wherein the antibody binds CD40. The antibody mayfurther include the heavy chain CDR2 and/or the light chain CDR2 ofantibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, or 3B6-NS. Theantibody may further comprise the heavy chain CDR1 and/or the lightchain CDR1 of antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, or3B6-NS.

Generation of Antibodies Having Modified Sequences

In another embodiment, the variable region sequences, or portionsthereof, of the anti-CD40 antibodies of the invention are modified tocreate structurally related anti-CD40 antibodies that retain binding(i.e., to the same epitope as the unmodified antibody) and, thus, arefunctionally equivalent. Methods for identifying residues that can bealtered without removing antigen binding are well-known in the art (see,e.g., Marks et al. (Biotechnology (1992) 10(7):779-83 (monoclonalantibodies diversification by shuffling light chain variable regions,then heavy chain variable regions with fixed CDR3 sequence changes),Jespers et al. (1994) Biotechnology 12(9):899-903 (selection of humanantibodies from phage display repertoires to a single epitope of anantigen), Sharon et al. (1986) PNAS USA 83(8):2628-31 (site-directedmutagenesis of an invariant amino acid residue at the variable-diversitysegments junction of an antibody); Casson et al. (1995) J. Immunol.155(12):5647-54 (evolution of loss and change of specificity resultingfrom random mutagenesis of an antibody heavy chain variable region).

Accordingly, in one aspect of the invention, the CDR1, 2, and/or 3regions of the engineered antibodies described above can comprise theexact amino acid sequence(s) as those of antibodies 3C3, 3G5, 1B4, 3B6,6H6, 2E1.2, 1B5-NK, or 3B6-NS disclosed herein. However, in otheraspects of the invention, the antibodies comprise derivatives from theexact CDR sequences of 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, or3B6-NS, yet still retain the ability of to bind CD40 effectively. Suchsequence modifications may include one or more amino acid additions,deletions, or substitutions, e.g., conservative sequence modificationsas described above. Sequence modifications may also be based on theconsensus sequences described above for the particular CDR1, CDR2, andCDR3 sequences of antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, or3B6-NS.

Accordingly, in another embodiment, the engineered antibody may becomposed of one or more CDRs that are, for example, 90%, 95%, 98% or99.5% identical to one or more CDRs of antibodies 3C3, 3G5, 1B4, 3B6,6H6, 2E1.2, 1B5-NK, or 3B6-NS. Ranges intermediate to the above-recitedvalues, e.g., CDRs that are 90-95%, 95-98%, or 98-100% identicalidentity to one or more of the above sequences are also intended to beencompassed by the present invention.

In another embodiment, one or more residues of a CDR may be altered tomodify binding to achieve a more favored on-rate of binding, a morefavored off-rate of binding, or both, such that an idealized bindingconstant is achieved. Using this strategy, an antibody having ultra highbinding affinity of, for example, 10¹⁰ M⁻¹ or more, can be achieved.Affinity maturation techniques, well known in the art and thosedescribed herein, can be used to alter the CDR region(s) followed byscreening of the resultant binding molecules for the desired change inbinding. Accordingly, as CDR(s) are altered, changes in binding affinityas well as immunogenicity can be monitored and scored such that anantibody optimized for the best combined binding and low immunogenicityare achieved.

In addition to or instead of modifications within the CDRs,modifications can also be made within one or more of the frameworkregions, FR1, FR2, FR3 and FR4, of the heavy and/or the light chainvariable regions of a antibody, so long as these modifications do noteliminate the binding affinity of the antibody. For example, one or morenon-germline amino acid residues in the framework regions of the heavyand/or the light chain variable region of a antibody of the invention,is substituted with a germline amino acid residue, i.e., thecorresponding amino acid residue in the human germline sequence for theheavy or the light chain variable region, which the antibody hassignificant sequence identity with. For example, an antibody chain canbe aligned to a germline antibody chain which it shares significantsequence identity with, and the amino acid residues which do not matchbetween antibody framework sequence and the germline chain framework canbe substituted with corresponding residues from the germline sequence.When an amino acid differs between a antibody variable framework regionand an equivalent human germline sequence variable framework region, theantibody framework amino acid should usually be substituted by theequivalent human germline sequence amino acid if it is reasonablyexpected that the amino acid falls within one of the followingcategories:

(1) an amino acid residue which noncovalently binds antigen directly,

(2) an amino acid residue which is adjacent to a CDR region,

(3) an amino acid residue which otherwise interacts with a CDR region(e.g., is within about 3-6 Å of a CDR region as determined by computermodeling), or

(4) an amino acid reside which participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which have a good probability ofdirectly interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, Van derWaals forces, hydrophobic interactions, and the like. Accordingly, inone embodiment, an amino acid residue in the framework region of aantibody of the invention is substituted with the corresponding germlineamino acid residue which noncovalently binds antigen directly.

Residues which are “adjacent to a CDR region” include amino acidresidues in positions immediately adjacent to one or more of the CDRs inthe primary sequence of the antibody, for example, in positionsimmediately adjacent to a CDR as defined by Kabat, or a CDR as definedby Chothia (see e.g., Chothia and Lesk J. Mol. Biol. 196:901 (1987)).Accordingly, in one embodiment, an amino acid residue within theframework region of an antibody of the invention is substituted with acorresponding germline amino acid residue which is adjacent to a CDRregion.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to affect a CDR region. Such amino acids willgenerally have a side chain atom within about 3 angstrom units (A) ofsome atom in the CDRs and must contain an atom that could interact withthe CDR atoms according to established chemical forces, such as thoselisted above. Accordingly, in one embodiment, an amino acid residuewithin the framework region of an antibody of the invention issubstituted with the corresponding germline amino acid residue whichotherwise interacts with a CDR region.

The amino acids at several positions in the framework are known to beimportant for determining CDR confirmation (e.g., capable of interactingwith the CDRs) in many antibodies (Chothia and Lesk, supra, Chothia etal., supra and Tramontano et al., J. Mol. Biol. 215:175 (1990), all ofwhich are incorporated herein by reference). These authors identifiedconserved framework residues important for CDR conformation by analysisof the structures of several known antibodies. The antibodies analyzedfell into a limited number of structural or “canonical” classes based onthe conformation of the CDRs. Conserved framework residues withinmembers of a canonical class are referred to as “canonical” residues.Canonical residues include residues 2, 25, 29, 30, 33, 48, 64, 71, 90,94 and 95 of the light chain and residues 24, 26, 29, 34, 54, 55, 71 and94 of the heavy chain. Additional residues (e.g., CDRstructure-determining residues) can be identified according to themethodology of Martin and Thorton (1996) J. Mol. Biol. 263:800. Notably,the amino acids at positions 2, 48, 64 and 71 of the light chain and26-30, 71 and 94 of the heavy chain (numbering according to Kabat) areknown to be capable of interacting with the CDRs in many antibodies. Theamino acids at positions 35 in the light chain and 93 and 103 in theheavy chain are also likely to interact with the CDRs. Additionalresidues which may effect conformation of the CDRs can be identifiedaccording to the methodology of Foote and Winter (1992) J. Mol. Biol.224:487. Such residues are termed “vernier” residues and are thoseresidues in the framework region closely underlying (i.e., forming a“platform” under) the CDRs.

Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA,82:4592-66 (1985) or Chothia et al, supra.

Occasionally, there is some ambiguity about whether a particular aminoacid falls within one or more of the above-mentioned categories. In suchinstances, alternative variant antibodies are produced, one of which hasthat particular substitution, the other of which does not. Alternativevariant antibodies so produced can be tested in any of the assaysdescribed herein for the desired activity, and the preferred antibodyselected.

Additional candidates for substitution within the framework region areamino acids that are unusual or “rare” for an antibody at that position.These amino acids can be substituted with amino acids from theequivalent position of the human germline sequence or from theequivalent positions of more typical antibodies. For example,substitution may be desirable when the amino acid in a framework regionof the antibody is rare for that position and the corresponding aminoacid in the germline sequence is common for that position inimmunoglobulin sequences; or when the amino acid in the antibody is rarefor that position and the corresponding amino acid in the germlinesequence is also rare, relative to other sequences. It is contemplatedthat by replacing an unusual amino acid with an amino acid from thegermline sequence that happens to be typical for antibodies, theantibody may be made less immunogenic. Substitution may also bedesirable, for example in cases of unpaired cysteine residues orputative N-linked glycosylation sites.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20%, preferably less than about 10%,more preferably less than about 5%, even more preferably less than about3%, even more preferably less than about 2% and even more preferablyless than about 1% of sequences in a representative sample of sequences,and the term “common”, as used herein, indicates an amino acid occurringin more than about 25% but usually more than about 50% of sequences in arepresentative sample. For example, all light and heavy chain variableregion sequences are respectively grouped into “subgroups” of sequencesthat are especially homologous to each other and have the same aminoacids at certain critical positions (Kabat et al., supra). When decidingwhether an amino acid in an antibody sequence is “rare” or “common”among sequences, it will often be preferable to consider only thosesequences in the same subgroup as the antibody sequence.

In general, the framework regions of antibodies are usuallysubstantially identical, and more usually, identical to the frameworkregions of the human germline sequences from which they were derived. Ofcourse, many of the amino acids in the framework region make little orno direct contribution to the specificity or affinity of an antibody.Thus, many individual conservative substitutions of framework residuescan be tolerated without appreciable change of the specificity oraffinity of the resulting immunoglobulin. Thus, in one embodiment thevariable framework region of the antibody shares at least 85% sequenceidentity to a human germline variable framework region sequence orconsensus of such sequences. In another embodiment, the variableframework region of the antibody shares at least 90%, 95%, 96%, 97%, 98%or 99% sequence identity to a human germline variable framework regionsequence or consensus of such sequences.

In addition to simply binding CD40, an antibody may be selected for itsretention of other functional properties of antibodies of the invention,such as, for example:

(a) inducing or enhancing an immune response to an antigen independentof Fc receptor binding;

(b) inducing or enhancing an immune response to an antigen withoutinducing antibody-dependent cellular cytotoxicity (ADCC) of CD40expressing cells;

(c) inducing or enhancingan immune response to an antigen withoutinducing complement dependent cellular cytotoxicity (CDC) of CD40expressing cells; and/or

(d) capable of synergising with CD40L.

Additional features may include, for example:

(e) no blocking of binding of CD40L to human CD40 independent of Fcreceptor binding;

(f) blocking of binding of CD40L to human CD40 independent of Fcreceptor binding;

(g) activation of human CD40 expressed on an APC, independent of Fcreceptor binding;

(h) induction of apoptosis of a tumor cell;

(i) T-cell stimulatory activity; and/or

(j) enhanced B-cell activation.

Characterization of Monoclonal Antibodies to CD40

Monoclonal antibodies of the invention can be characterized for bindingto CD40 using a variety of known techniques. Generally, the antibodiesare initially characterized by ELISA. Briefly, microtiter plates can becoated with purified CD40 in PBS, and then blocked with irrelevantproteins such as bovine serum albumin (BSA) diluted in PBS. Dilutions ofplasma from CD40-immunized mice are added to each well and incubated for1-2 hours at 37° C. The plates are washed with PBS/Tween 20 and thenincubated with a goat-anti-human IgG Fc-specific polyclonal reagentconjugated to alkaline phosphatase for 1 hour at 37° C. After washing,the plates are developed with ABTS substrate, and analyzed at OD of 405.Preferably, mice which develop the highest titers will be used forfusions.

An ELISA assay as described above can be used to screen for antibodiesand, thus, hybridomas that produce antibodies that show positivereactivity with the CD40 immunogen. Hybridomas that bind, preferablywith high affinity, to CD40 can then be subcloned and furthercharacterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can then be chosen for makinga cell bank, and for antibody purification.

To purify anti-CD40 antibodies, selected hybridomas can be grown inroller bottles, two-liter spinner-flasks or other culture systems.Supernatants can be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.) topurify the protein. After buffer exchange to PBS, the concentration canbe determined by OD₂₈₀ using 1.43 extinction coefficient or preferablyby nephelometric analysis. IgG can be checked by gel electrophoresis andby antigen specific method.

To determine if the selected anti-CD40 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Biotinylated MAb bindingcan be detected with a streptavidin labeled probe. To determine theisotype of purified antibodies, isotype ELISAs can be performed usingart recognized techniques. For example, wells of microtiter plates canbe coated with 10 μg/ml of anti-Ig overnight at 4° C. After blockingwith 5% BSA, the plates are reacted with 10 μg/ml of monoclonalantibodies or purified isotype controls, at ambient temperature for twohours. The wells can then be reacted with either IgG or other isotypespecific conjugated probes. Plates are developed and analyzed asdescribed above.

To test the binding of monoclonal antibodies to live cells expressingCD40, flow cytometry can be used. Briefly, cell lines and/or human PBMCsexpressing membrane-bound CD40 (grown under standard growth conditions)are mixed with various concentrations of monoclonal antibodies in PBScontaining 0.1% BSA at 4° C. for 1 hour. After washing, the cells arereacted with Fluorescein-labeled anti-IgG antibody under the sameconditions as the primary antibody staining. The samples can be analyzedby FACScan instrument using light and side scatter properties to gate onsingle cells and binding of the labeled antibodies is determined. Analternative assay using fluorescence microscopy may be used (in additionto or instead of) the flow cytometry assay. Cells can be stained exactlyas described above and examined by fluorescence microscopy. This methodallows visualization of individual cells, but may have diminishedsensitivity depending on the density of the antigen.

Anti-CD40 IgGs can be further tested for reactivity with the CD40antigen by Western blotting. Briefly, cell extracts from cellsexpressing CD40 can be prepared and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens will be transferred to nitrocellulose membranes, blocked with20% mouse serum, and probed with the monoclonal antibodies to be tested.IgG binding can be detected using anti-IgG alkaline phosphatase anddeveloped with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis,Mo.).

Methods for analyzing binding affinity, cross-reactivity, and bindingkinetics of various anti-CD40 antibodies include standard assays knownin the art, for example, Biacore™ surface plasmon resonance (SPR)analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala,Sweden), or bio-layer interferometry (BLI) using an Octet™ QK^(e)instrument as described in the examples.

Agonistic anti-CD40 antibodies which bind to the same epitope as that ofanti-CD40 antibodies 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, and 3B6-NS(as determined by a given epitope mapping technique) also are providedherein. For example, as described in Example 17, antibodies of theinvention (e.g., antibody 3C3) bind to one or more residues within aminoacid residues 1-5 and 33-36 of the extracellular domain (ECD) of humanCD40 (SEQ ID NO: 133), e.g., amino acids 5, 33, 34 and/or 36 of the ECDof human CD40 (SEQ ID NO: 133). Antibody 3C3 also is shown to furtherbind to one or more amino acids 26, 28 and/or 30 of the ECD of humanCD40 (SEQ ID NO: 133), e.g., amino acids 5, 33, 34 and 36 of the ECD ofhuman CD40 (SEQ ID NO: 133) or amino acids 5, 33 and 36 of the ECD ofhuman CD40 (SEQ ID NO: 133).

Other antibodies of the invention (e.g., antibody 3G5) bind to one ormore residues within amino acid residues 13-15 and 33-36 of the ECD ofhuman CD40 (SEQ ID NO: 133), e.g., amino acids 33, 34 and 36 of the ECDof human CD40 (SEQ ID NO: 133).

Antibodies which bind to the epitopes on human CD40 described herein(e.g., the same epitopes as the exemplified antibodies) exhibittherapeutically advantageous properties. For example, as demonstrated inExamples 16 and 20, antibody 3C3 exhibits synergistic agnostic effectswith soluble CD40 ligand (sCD40L), as measured by, for example, anincrease in the induction of CD95 expression when incubated with Ramoscells, an increase in B cell proliferation when incubated with human Bcells, and/or an increase in the induction of IL12p40 expression whenincubated with dendritic cells.

Accordingly, antibodies that bind to the same epitope as 3C3 have theability to synergize with other therapeutic agents, including thosewhich bind to the ligand binding site of human CD40. Representativesynergistic effects include, for example, upregulation of immunefunction (e.g. T cell mediated immune responses as in vaccine therapies,NK activation in cancer therapies), inhibition of cell growth (e.g., incancer therapy), and/or enhanced processing and presentation of anantigen by APCs (e.g., in vaccine therapy).

As described herein, techniques for determining antibodies that bind tothe “same epitope on CD40” with the antibodies described herein include,for example, epitope mapping methods, such as, x-ray analyses ofcrystals of antigen:antibody complexes which provides atomic resolutionof the epitope. Other methods monitor the binding of the antibody toantigen fragments or mutated variations of the antigen where loss ofbinding due to a modification of an amino acid residue within theantigen sequence is often considered an indication of an epitopecomponent. In addition, computational combinatorial methods for epitopemapping can also be used. Methods may also rely on the ability of anantibody of interest to affinity isolate specific short peptides (eitherin native three dimensional form or in denatured form) fromcombinatorial phage display peptide libraries. The peptides are thenregarded as leads for the definition of the epitope corresponding to theantibody used to screen the peptide library. For epitope mapping,computational algorithms have also been developed which have been shownto map conformational discontinuous epitopes.

II. Molecular Conjugates/Immunotoxins

The present invention provides a variety of therapeutic molecularconjugates (e.g., vaccine conjugates) which include an antigen, such asa tumor or viral antigen, linked to an antibody that binds to a receptoron an APC, for example, an antibody which binds to CD40. This allows fortargeting of the antigen to APCs, such as cells expressing CD40 (e.g.,dendritic cells, B cells, and macrophages) to enhance processing,presentation and, ultimately, an immune response against the antigen(s).A schematic representation of such a conjugate is shown in FIG. 18wherein, for example, an antigen is genetically fused to the CH3 domainof each of the heavy chains of a substantially complete anti-CD40antibody. However, it will be appreciated that the antigen mayalternatively be joined to other parts of such an antibody or fragmentthereof, and that other forms of conjugation, such as chemicalconjugation, may also be employed, as discussed further below.

Suitable antigens for use in the molecular conjugates include, forexample, infectious disease antigens and tumor antigens, against whichprotective or therapeutic immune responses are desired, e.g., antigensexpressed by a tumor cell or a pathogenic organism or infectious diseaseantigens. For example, suitable antigens include tumor-associatedantigens for the prevention or treatment of cancers. Examples oftumor-associated antigens include, but are not limited to, sequencescomprising all or part of the sequences of βhCG, gpl00 or Pmel17,HER2/neu, WT1, mesothelin, CEA, gpl00, MART1, TRP-2, melan-A, NY-ESO-1,NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MAGE-3, MAGE-A3,Tyrosinase, Telomerase, SSX2 and MUC-1 antigens, and germ cell derivedtumor antigens. Tumor associated antigens also include the blood groupantigens, for example, Le^(a), Le^(b), LeX, LeY, H-2, B-1, B-2 antigens.Alternatively, more than one antigen can be included within theantigen-antibody constructs of the invention. For example, a MAGEantigen can be combined with other antigens such as melanin A,tyrosinase, and gpl00 along with adjuvants such as GM-CSF or IL-12, andlinked to an anti-APC antibody.

Other suitable antigens include viral antigens for the prevention ortreatment of viral diseases. Examples of viral antigens include, but arenot limited to, HIV-1 gag, HIV-1 env, HIV-1 nef, HBV (surface or coreantigens), HPV, FAS, HSV-1, HSV-2, p17, ORF2 and ORF3 antigens. Examplesof bacterial antigens include, but are not limited to, Toxoplasma gondiior Treponema pallidum. The antibody-bacterial antigen conjugates of theinvention can be in the treatment or prevention of various bacterialdiseases such as Anthrax, Botulism, Tetanus, Chlamydia, Cholera,Diphtheria, Lyme Disease, Syphilis and Tuberculosis.

Sequences of the above-described antigens are well known in the art. Forexample, an example of a MAGE-3 cDNA sequence is provided in U.S. Pat.No. 6,235,525 (Ludwig Institute for Cancer Research); examples ofNY-ESO-1 nucleic acid and protein sequences are provided in U.S. Pat.Nos. 5,804,381 and 6,069,233 (Ludwig Institute for Cancer Research);examples of Melan-A nucleic acid and protein sequences are provided inU.S. Pat. Nos. 5,620,886 and 5,854,203 (Ludwig Institute for CancerResearch); examples of NY-BR-1 nucleic acid and protein sequences areprovided in U.S. Pat. Nos. 6,774,226 and 6,911,529 (Ludwig Institute forCancer Research) and examples of NY-CO-58 nucleic acid and proteinsequences are provided in WO 02090986 (Ludwig Institute for CancerResearch); an example of an amino acid sequence for the HER-2/neuprotein is available at GENBANK® Accession No. AAA58637; and anucleotide sequence (mRNA) for human carcinoembryonic antigen-like 1(CEA-1) is available at GENBANK® Accession No. NM__020219.

In one embodiment, the antigen is an HPV antigen, for example, HPV-16antigen, an HPV-18 antigen, an HPV-31 antigen, an HPV-33 antigen and/orHPV-35 antigen. A genome of HPV-16 is described in Virology, 145:181-185(1985) and DNA sequences encoding HPV-18 are described in U.S. Pat. No.5,840,306, the disclosures of which are incorporated by reference hereinin their entirety. HIPV-16 antigens (e.g., seroreactive regions of theE1 and/or E2 proteins of HPV-16) are described in U.S. Pat. No.6,531,127, and HPV-18 antigens (e.g., seroreactive regions of the L1and/or L2 proteins of HPV-18) are described in U.S. Pat. No. 5,840,306,the disclosures of which are incorporated by reference herein.Similarly, a complete genome for HBV is available at GENBANK® AccessionNo. NC_003977, the disclosure of which is incorporated herein. Thegenome of HCV is described in European Patent Application No. 318 216,the disclosure of which is incorporated herein. PCT/US90/01348,incorporated by reference herein, discloses sequence information ofclones of the HCV genome, amino acid sequences of HCV viral proteins andmethods of making and using such compositions for HCV vaccinescomprising HCV proteins and peptides derived therefrom.

Antigenic peptides of proteins (i.e., those containing T cell epitopes)can be identified in a variety of manners well known in the art. Forexample, T cell epitopes can be predicted by analyzing the sequence ofthe protein using web-based predictive algorithms (BIMAS & SYFPEITHI) togenerate potential MHC class I and II-binding peptides that match aninternal database of 10,000 well characterized MHC binding peptidespreviously defined by CTLs. High scoring peptides can be ranked andselected as “interesting” on the basis of high affinity to a given MHCmolecule.

Another method for identifying antigenic peptides containing T cellepitopes involves dividing the protein into non-overlapping peptides ofdesired length or overlapping peptides of desired lengths which can beproduced recombinantly, synthetically, or in certain limited situations,by chemical cleavage of the protein and tested for immunogenicproperties, e.g., eliciting a T cell response (i.e., proliferation orlymphokine secretion).

In order to determine precise T cell epitopes of the protein by, forexample, fine mapping techniques, a peptide having T cell stimulatingactivity and thus comprising at least one T cell epitope, as determinedby T cell biology techniques, can be modified by addition or deletion ofamino acid residues at either the amino or carboxy terminus of thepeptide and tested to determine a change in T cell reactivity to themodified peptide. If two or more peptides which share an area of overlapin the native protein sequence are found to have human T cellstimulating activity, as determined by T cell biology techniques,additional peptides can be produced comprising all or a portion of suchpeptides and these additional peptides can be tested by a similarprocedure. Following this technique, peptides are selected and producedrecombinantly or synthetically. Peptides are selected based on variousfactors, including the strength of the T cell response to the peptide(e.g., stimulation index). The physical and chemical properties of theseselected peptides (e.g., solubility, stability) can then be examined todetermine whether the peptides are suitable for use in therapeuticcompositions or whether the peptides require modification.

In addition, the vaccine conjugate can include one or moreimmunostimulatory agents that also enhance the immune response againstthe antigen. Antibody-antigen vaccine conjugates of the invention can bemade genetically or chemically. In either case, the antibody portion ofthe conjugate may consist of the whole antibody or a portion of theantibody, such as the Fab fragment or single-chain Fv. In addition, morethan one antigen and/or immunostimulatory agent can be included in theconjugate.

Chemically constructed antibody-antigen conjugates can be made using avariety of well known and readily available cross-linking reagents.These cross-linking reagents can be homofunctional or heterofunctionalcompounds, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl-S-acetyl-thioacetate (SATA), sulfosuccinimidyl4-(N-maleimidomethyl) cyclohaxane-1-carboxylate (sulfo-SMCC),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), that form covalent linkageswith different reactive amino acid or carbohydrate side chains on theanti-dendritic antibody and selected antigen. Other coupling andcross-linking agents also can be used to generate covalent linkages,such as protein A, carbodiimide, and o-phenylenedimaleimide (oPDM); (seee.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, M A et al.(1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include thosedescribed by Paulus (Behring Ins. Mitt. (1985) No. 78, 118-132); Brennanet al. (Science (1985) 229:81-83), and Glennie et al. (J. Immunol.(1987) 139: 2367-2375). Preferred conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).Immunostimulatory agents can also be chemically linked to the molecularconjugates of the present invention using the same linking methodsdescribed above.

In another embodiment, the antibodies of the present invention arelinked to a therapeutic moiety, such as a cytotoxin, a drug or aradioisotope. When conjugated to a cytotoxin, these antibody conjugatesare referred to as “immunotoxins.” A cytotoxin or cytotoxic agentincludes any agent that is detrimental to (e.g., kills) cells. Examplesinclude taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). An antibody of the presentinvention can be conjugated to a radioisotope, e.g., radioactive iodine,to generate cytotoxic radiopharmaceuticals for treating adendritic-related disorder, such as an autoimmune or inflammatorydisease, or graft versus host disease.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

III. Compositions

In another embodiment, the present invention provides a composition,e.g., a composition, containing one or a combination of monoclonalantibodies of the present invention, formulated together with a carrier(e.g., a pharmaceutically acceptable carrier). Compositions containingbispecific molecules which comprise an antibody of the present inventionare also provided. In one embodiment, the compositions include acombination of multiple (e.g., two or more) isolated antibodies of theinvention. Preferably, each of the antibodies of the composition bindsto a distinct, pre-selected epitope of CD40.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition of the present inventionwith at least one or more additional therapeutic agents, such asanti-inflammatory agents, DMARDs (disease-modifying anti-rheumaticdrugs), immunosuppressive agents, and chemotherapeutics. Thepharmaceutical compositions of the invention can also be administered inconjunction with radiation therapy. Co-administration with otherantibodies is also encompassed by the invention.

As used herein, the terms “carrier” and “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Preferably,the carrier is suitable for intravenous, intramuscular, subcutaneous,parenteral, spinal or epidermal administration (e.g., by injection orinfusion). Depending on the route of administration, the activecompound, i.e., antibody, bispecific and multispecific molecule, may becoated in a material to protect the compound from the action of acidsand other natural conditions that may inactivate the compound.

Examples of adjuvants which may be used with the antibodies andconstructs of the present invention include: Freund's IncompleteAdjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.);Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatised polysaccharides;polyphosphazenes; biodegradable microspheres; cytokines, such as GM-CSF,interleukin-2, -7, -12, and other like factors; 3D-MPL; CpGoligonucleotide; and monophosphoryl lipid A, for example 3-de-O-acylatedmonophosphoryl lipid A.

MPL adjuvants are available from Corixa Corporation (Seattle, Wash.;see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and4,912,094). CpG-containing oligonucleotides (in which the CpGdinucleotide is unmethylated) are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996.

Further alternative adjuvants include, for example, saponins, such asQuil A, or derivatives thereof, including QS21 and QS7 (AquilaBiopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; orGypsophila or Chenopodium quinoa saponins; Montanide ISA 720 (Seppic,France); SAF (Chiron, Calif., United States); ISCOMS (CSL), MF-59(Chiron); the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium); Detox(Enhanzyn™) (Corixa, Hamilton, Mont.); RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs); polyoxyethyleneether adjuvants such as those described in WO 99/52549A1; syntheticimidazoquinolines such as imiquimod [S-26308, R-837], (Harrison, et al.,Vaccine 19: 1820-1826, 2001; and resiquimod [S-28463, R-848] (Vasilakos,et al., Cellular immunology 204: 64-74, 2000; Schiff bases of carbonylsand amines that are constitutively expressed on antigen presenting celland T-cell surfaces, such as tucaresol (Rhodes, J. et al., Nature 377:71-75, 1995); cytokine, chemokine and co-stimulatory molecules as eitherprotein or peptide, including for example pro-inflammatory cytokinessuch as Interferon, GM-CSF, IL-1 alpha, IL-1 beta, TGF-alpha andTGF-beta, Th1 inducers such as interferon gamma, IL-2, IL-12, IL-15,IL-18 and IL-21, Th2 inducers such as IL-4, IL-5, IL-6, IL-10 and IL-13and other chemokine and co-stimulatory genes such as MCP-1, MIP-1 alpha,MIP-1 beta, RANTES, TCA-3, CD80, CD86 and CD70; immunostimulatory agentstargeting ligands such as CTLA-4 and L-selectin, apoptosis stimulatingproteins and peptides such as Fas; synthetic lipid based adjuvants, suchas vaxfectin, (Reyes et al., Vaccine 19: 3778-3786, 2001) squalene,alpha-tocopherol, polysorbate 80, DOPC and cholesterol; endotoxin,[LPS], (Beutler, B., Current Opinion in Microbiology 3: 23-30, 2000);ligands that trigger Toll receptors to produce Th1-inducing cytokines,such as synthetic Mycobacterial lipoproteins, Mycobacterial protein p19,peptidoglycan, teichoic acid and lipid A; and CT (cholera toxin,subunits A and B) and LT (heat labile enterotoxin from E. coli, subunitsA and B), heat shock protein family (HSPs), and LLO (listeriolysin O; WO01/72329). These and various further Toll-like Receptor (TLR) agonistsare described for example in Kanzler et al, Nature Medicine, May 2007,Vol 13, No 5. A preferred immunostimulatory agent for use in combinationwith an anti-CD40 antibody of the invention is a TLR3 agonist, such asPoly IC.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent. Acceptablediluents include saline and aqueous buffer solutions. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al. (1984) J. Neuroimmunol. 7:27).

Carriers include sterile aqueous solutions or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. The use of such media and agents forpharmaceutically active substances is known in the art. Except insofaras any conventional media or agent is incompatible with the activecompound, use thereof in the pharmaceutical compositions of theinvention is contemplated. Supplementary active compounds can also beincorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. For example, the antibodies ofthe invention may be administered once or twice weekly by subcutaneousor intramuscular injection or once or twice monthly by subcutaneous orintramuscular injection.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of one hundredpercent, this amount will range from about 0.001 percent to about ninetypercent of active ingredient, preferably from about 0.005 percent toabout 70 percent, most preferably from about 0.01 percent to about 30percent.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.001 to 90% (morepreferably, 0.005 to 70%, such as 0.01 to 30%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. A physician orveterinarian having ordinary skill in the art can readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician or veterinarian could start dosesof the compounds of the invention employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose of acomposition of the invention will be that amount of the compound whichis the lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.It is preferred that administration be intravenous, intramuscular,intraperitoneal, or subcutaneous, preferably administered proximal tothe site of the target. If desired, the effective daily dose of atherapeutic composition may be administered as two, three, four, five,six or more sub-doses administered separately at appropriate intervalsthroughout the day, optionally, in unit dosage forms. While it ispossible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824,or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known to those skilledin the art.

In certain embodiments, the antibodies of the invention can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds of the invention cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134),different species of which may comprise the formulations of theinventions, as well as components of the invented molecules; p120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273. In one embodiment of the invention, thetherapeutic compounds of the invention are formulated in liposomes; in amore preferred embodiment, the liposomes include a targeting moiety. Ina most preferred embodiment, the therapeutic compounds in the liposomesare delivered by bolus injection to a site proximal to the tumor orinfection. The composition must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi.

The ability of a compound to inhibit cancer can be evaluated in ananimal model system predictive of efficacy in human tumors.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit, such inhibition invitro by assays known to the skilled practitioner. A therapeuticallyeffective amount of a therapeutic compound can decrease tumor size, orotherwise ameliorate symptoms in a subject. One of ordinary skill in theart would be able to determine such amounts based on such factors as thesubject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

IV. Uses and Methods of the Invention

Antibodies, molecular conjugates, bispecific molecules, and compositionsof the present invention can be used to treat and/or prevent (e.g.,immunize against) a variety of diseases and conditions.

One of the primary disease indications is cancer. Types of cancersinclude, but are not limited to, leukemia, acute lymphocytic leukemia,acute myelocytic leukemia, myeloblasts promyelocyte myelomonocyticmonocytic erythroleukemia, chronic leukemia, chronic myelocytic(granulocytic) leukemia, chronic lymphocytic leukemia, mantle celllymphoma, primary central nervous system lymphoma, Burkitt's lymphomaand marginal zone B cell lymphoma, Polycythemia vera Lymphoma, Hodgkin'sdisease, non-Hodgkin's disease, multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, solid tumors, sarcomas, andcarcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma,osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, uterine cancer, testicular tumor, lung carcinoma, small celllung carcinoma, non small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basalcell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brainand central nervous system (CNS) cancer, cervical cancer,choriocarcinoma, colorectal cancers, connective tissue cancer, cancer ofthe digestive system, endometrial cancer, esophageal cancer, eye cancer,head and neck cancer, gastric cancer, intraepithelial neoplasm, kidneycancer, larynx cancer, liver cancer, lung cancer (small cell, largecell), melanoma, neuroblastoma; oral cavity cancer (for example lip,tongue, mouth and pharynx), ovarian cancer, pancreatic cancer,retinoblastoma, rhabdomyosarcoma, rectal cancer; cancer of therespiratory system, sarcoma, skin cancer, stomach cancer, testicularcancer, thyroid cancer, uterine cancer, and cancer of the urinarysystem. Particular cancers include CD40-expressing tumors selected fromthe group consisting of chronic lymphocytic leukemia, mantle celllymphoma, primary central nervous system lymphoma, Burkitt's lymphomaand marginal zone B cell lymphoma.

Antibodies and conjugates of the invention also can be used to treatbacterial, fungal, viral and parasitic infectious diseases.

When used in therapy, the antibodies of the invention can beadministered to a subject directly (i.e., in vivo), either alone or withother therapies such as an immunostimulatory agent, a vaccine,chemotherapy or radiation therapy. In all cases, the antibodies,conjugates, bispecifics, compositions, and immunostimulatory agents andother therapies are administered in an effective amount to exert theirdesired therapeutic effect. The term “effective amount” refers to thatamount necessary or sufficient to realize a desired biologic effect. Forexample, an effective amount could be that amount necessary to eliminatea tumor, cancer, or bacterial, viral or fungal infection. The effectiveamount for any particular application can vary depending on such factorsas the disease or condition being treated, the particular antibody beingadministered, the size of the subject, or the severity of the disease orcondition. One of ordinary skill in the art can empirically determinethe effective amount of a particular molecule without necessitatingundue experimentation.

Preferred routes of administration include, for example, injection(e.g., subcutaneous, intravenous, parenteral, intraperitoneal,intrathecal). The injection can be in a bolus or a continuous infusion.Other routes of administration include oral administration.

In another embodiment, the antibody is administered in combination witha vaccine antigen, to enhance the immune response against the vaccineantigen, such as a tumor antigen (to thereby enhance the immune responseagainst the tumor) or an antigen from an infectious disease pathogen (tothereby enhance the immune response against the infectious diseasepathogen). The vaccine antigen can be any antigen or antigeniccomposition capable of eliciting an immune response against a tumor oragainst an infectious disease pathogen such as a virus, a bacteria, aparasite or a fungus. It may also be, for example, a neoantigen such asthose derived from sequencing of patients' tumors. The antigen orantigens can be, for example, peptides/proteins, polysaccharides and/orlipids, or may be administered as nucleic acids (such as DNA) coding forpeptide or protein antigens which may be expressed in vivo. The antigenor antigens be derived from tumors, such as the various tumor antigenspreviously disclosed herein. Alternatively, the antigen or antigens canbe derived from pathogens such as viruses, bacteria, parasites and/orfungi, such as the various pathogen antigens previously disclosedherein. Additional examples of suitable pathogen antigens include, butare not limited to, the following:

Viral antigens or antigenic determinants can be derived from, forexample: Cytomegalovirus (especially Human, such as gB or derivativesthereof); Epstein Barr virus (such as gp350); flaviviruses (e.g. YellowFever Virus, Dengue Virus, Tick-borne encephalitis virus, JapaneseEncephalitis Virus); hepatitis virus such as hepatitis B virus (forexample Hepatitis B Surface antigen such as the PreS1, PreS2 and Santigens described in EP-A-414 374; EP-A-0304 578, and EP-A-198474),hepatitis A virus, hepatitis C virus and hepatitis E virus; HIV-1, (suchas tat, nef, gpl20 or gpl60); human herpes viruses, such as gD orderivatives thereof or Immediate Early protein such as ICP27 from HSV1or HSV2; human papilloma viruses (for example HPV6, 11, 16, 18);Influenza virus (whole live or inactivated virus, split influenza virus,grown in eggs or MDCK cells, or Vero cells or whole flu virosomes (asdescribed by Gluck, Vaccine, 1992, 10, 915-920) or purified orrecombinant proteins thereof, such as NP, NA, HA, or M proteins);measles virus; mumps virus; parainfluenza virus; rabies virus;Respiratory Syncytial virus (such as F and G proteins); rotavirus(including live attenuated viruses); smallpox virus; Varicella ZosterVirus (such as gpI, II and IE63); and the HPV viruses responsible forcervical cancer (for example the early proteins E6 or E7 in fusion witha protein D carrier to form Protein D-E6 or E7 fusions from HPV 16, orcombinations thereof, or combinations of E6 or E7 with L2 (see forexample WO 96/26277).

Bacterial antigens or antigenic determinants can be derived from, forexample: Bacillus spp., including B. anthracis (e.g., botulinum toxin);Bordetella spp, including B. pertussis (for example pertactin, pertussistoxin, filamenteous hemagglutinin, adenylate cyclase, fimbriae);Borrelia spp., including B. burgdorferi (eg OspA, OspC, DbpA, DbpB), B.garinii (eg OspA, OspC, DbpA, DbpB), B. afzelii (eg OspA, OspC, DbpA,DbpB), B. andersonii (eg OspA, OspC, DbpA, DbpB), B. hermsii;Campylobacter spp, including C. jejuni (for example toxins, adhesins andinvasins) and C. coli; Chlamydia spp., including C. trachomatis (egMOMP, heparin-binding proteins), C. pneumonie (eg MOMP, heparin-bindingproteins), C. psittaci; Clostridium spp., including C. tetani (such astetanus toxin), C. botulinum (for example botulinum toxin), C. difficile(eg clostridium toxins A or B); Corynebacterium spp., including C.diphtheriae (eg diphtheria toxin); Ehrlichia spp., including E. equi andthe agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp,including R. rickettsii; Enterococcus spp., including E. faecalis, E.faecium; Escherichia spp, including enterotoxic E. coli (for examplecolonization factors, heat-labile toxin or derivatives thereof, orheat-stable toxin), enterohemorragic E. coli, enteropathogenic E. coli(for example shiga toxin-like toxin); Haemophilus spp., including H.influenzae type B (eg PRP), non-typable H. influenzae, for exampleOMP26, high molecular weight adhesins, P5, P6, protein D and lipoproteinD, and fimbrin and fimbrin derived peptides (see for example U.S. Pat.No. 5,843,464); Helicobacter spp, including H. pylori (for exampleurease, catalase, vacuolating toxin); Pseudomonas spp, including P.aeruginosa; Legionella spp, including L. pneumophila; Leptospira spp.,including L. interrogans; Listeria spp., including L. monocytogenes;Moraxella spp, including M. catarrhalis, also known as Branhamellacatarrhalis (for example high and low molecular weight adhesins andinvasins); Morexella Catarrhalis (including outer membrane vesiclesthereof, and OMP106 (see for example WO97/41731)); Mycobacterium spp.,including M. tuberculosis (for example ESAT6, Antigen 85A, -B or -C), M.bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Neisseriaspp, including N. gonorrhea and N. meningitidis (for example capsularpolysaccharides and conjugates thereof, transferrin-binding proteins,lactoferrin binding proteins, PilC, adhesins); Neisseria mengitidis B(including outer membrane vesicles thereof, and NspA (see for example WO96/29412); Salmonella spp, including S. typhi, S. paratyphi, S.choleraesuis, S. enteritidis; Shigella spp, including S. sonnei, S.dysenteriae, S. flexnerii; Staphylococcus spp., including S. aureus, S.epidermidis; Streptococcus spp, including S. pneumonie (eg capsularpolysaccharides and conjugates thereof, PsaA, PspA, streptolysin,choline-binding proteins) and the protein antigen Pneumolysin (BiochemBiophys Acta, 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25,337-342), and mutant detoxified derivatives thereof (see for example WO90/06951; WO 99/03884); Treponema spp., including T. pallidum (eg theouter membrane proteins), T. denticola, T. hyodysenteriae; Vibrio spp,including V. cholera (for example cholera toxin); and Yersinia spp,including Y. enterocolitica (for example a Yop protein), Y. pestis, Y.pseudotuberculosis.

Parasitic/fungal antigens or antigenic determinants can be derived from,for example: Babesia spp., including B. microti; Candida spp., includingC. albicans; Cryptococcus spp., including C. neoformans; Entamoeba spp.,including E. histolytica; Giardia spp., including; G. lamblia; Leshmaniaspp., including L. major; Plasmodium. faciparum (MSP1, AMA1, MSP3, EBA,GLURP, RAP1, RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA,PfEXPI, Pfs25, Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and theiranalogues in Plasmodium spp.); Pneumocystis spp., including P. carinii;Schisostoma spp., including S. mansoni; Trichomonas spp., including T.vaginalis; Toxoplasma spp., including T. gondii (for example SAG2, SAG3,Tg34); Trypanosoma spp., including T. cruzi.

It will be appreciated that in accordance with this aspect of thepresent invention, antigens and antigenic determinants can be used inmany different forms. For example, antigens or antigenic determinantscan be present as isolated proteins or peptides (for example inso-called “subunit vaccines”) or, for example, as cell-associated orvirus-associated antigens or antigenic determinants (for example ineither live or killed pathogen strains). Live pathogens will preferablybe attenuated in known manner. Alternatively, antigens or antigenicdeterminants may be generated in situ in the subject by use of apolynucleotide coding for an antigen or antigenic determinant (as inso-called “DNA vaccination”), although it will be appreciated that thepolynucleotides which can be used with this approach are not limited toDNA, and may also include RNA and modified polynucleotides as discussedabove.

When used in therapy, molecular conjugates (i.e., vaccine conjugates) ofthe invention can be administered to a subject directly (i.e., in vivo),either alone or with an immunostimulatory agent. In one aspect, theimmunostimulatory agent is linked to the conjugate. Alternatively, theconjugates can be administered to a subject indirectly by firstcontacting the conjugates (e.g., by culturing or incubating) with APCs,such as dendritic cells, and then administering the cells to the subject(i.e., ex vivo). The contacting and delivering of the conjugates toAPCs, such that they are processed and presented by the APCs prior toadministration, is also referred to as antigen or cell “loading.”Techniques for loading antigens to APCs are well known in the art andinclude, for example, Gunzer and Grabbe, Crit Rev Immunol 21(1-3):133-45 (2001) and Steinman, Exp Hematol 24(8): 859-62 (1996).

In all cases, the vaccine conjugates and the immunostimulatory agentsare administered in an effective amount to exert their desiredtherapeutic effect.

Antibodies, molecular conjugates, bispecific molecules, and compositionsof the invention also can be coadministered with adjuvants and othertherapeutic agents. It will be appreciated that the term“coadministered” as used herein includes any or all of simultaneous,separate, or sequential administration of the antibodies and conjugatesof the present invention with adjuvants and other agents, includingadministration as part of a dosing regimen. The antibodies are typicallyformulated in a carrier alone or in combination with such agents.Examples of such carriers include solutions, solvents, dispersion media,delay agents, emulsions and the like. The use of such media forpharmaceutically active substances is well known in the art. Any otherconventional carrier suitable for use with the molecules falls withinthe scope of the instant invention.

Suitable agents for co-administration with the antibodies, conjugates,bispecifics, and compositions include other antibodies, cytotoxinsand/or drugs, as well as adjuvants, immunostimulatory agents and/orimmunosuppressive agents. In one embodiment, the agent is achemotherapeutic agent. The antibodies, bispecifics, and compositionscan be administered in combination with radiation.

Chemotherapeutic agents suitable for coadministration with theantibodies and conjugates of the present invention in the treatment oftumors include, for example: taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Furtheragents include, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine) and temozolomide.

Agents that delete or inhibit immunosuppressive activities, for example,by immune cells (for example regulatory T-cells, NKT cells, macrophages,myeloid-derived suppressor cells, immature or suppressive dendriticcells) or suppressive factors produced by the tumor or host cells in thelocal microenvironment of the tumor (for example, TGFbeta, indoleamine2,3 dioxygenase—IDO), may also be administered with the antibodies andconjugates of the present invention. Such agents include antibodies andsmall molecule drugs such as IDO inhibitors such as 1 methyl tryptophanor derivatives.

Suitable agents for coadministration with the antibodies, conjugates,and bispecifics of the present invention for inducement or enhancementof an immune response include, for example, adjuvants and/orimmunostimulatory agents, non-limiting examples of which have beendisclosed hereinbefore. A preferred immunostimulatory agent is a TLR3agonist, such as Poly IC.

V. Combination Therapies

The anti-CD40 antibodies described herein also can be used incombination therapy, e.g., for treating cancer. Accordingly, providedherein are methods of combination therapy in which an anti-CD40 antibodyis co-administered with one or more additional agents, e.g., smallmolecule drugs, antibodies or antigen binding portions thereof, and/orprotein ligands that are effective in stimulating immune responses tothereby further enhance, stimulate or upregulate immune responses in asubject. Moreover, as shown in the Examples herein, administration of anagonist anti-CD40 antibody and soluble CD40 ligand had a synergic effectin inducing T cell receptor-mediated signals, e.g., as shown by theincrease in the expression of CD95 in tumor cells.

For example, an anti-CD40 antibody, e.g., described herein, can becombined with (i) an agonist of a stimulatory (e.g., co-stimulatory)molecule (e.g., receptor or ligand) and/or (ii) an antagonist of aninhibitory signal or molecule (e.g., receptor or ligand) on immunecells, such as T cells, both of which result in amplifying immuneresponses, such as antigen-specific T cell responses. In certainaspects, an immuno-oncology agent is (i) an agonist of a stimulatory(including a co-stimulatory) molecule (e.g., receptor or ligand) or (ii)an antagonist of an inhibitory (including a co-inhibitory) molecule(e.g., receptor or ligand) on cells involved in innate immunity, e.g.,NK cells, and wherein the immuno-oncology agent enhances innateimmunity. Such immuno-oncology agents are often referred to as immunecheckpoint regulators, e.g., immune checkpoint inhibitor or immunecheckpoint stimulator.

In one embodiment, an anti-CD40 antibody is administered with an agentthat targets a stimulatory or inhibitory molecule that is a member ofthe immunoglobulin super family (IgSF). For example, anti-CD40antibodies, e.g., described herein, may be administered to a subjectwith an agent that targets a member of the IgSF family to increase animmune response. For example, an anti-CD40 antibody may be administeredwith an agent that targets (or binds specifically to) a member of the B7family of membrane-bound ligands that includes B7-1, B7-2, B7-H1(PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), andB7-H6 or a co-stimulatory or co-inhibitory receptor binding specificallyto a B7 family member.

An anti-CD40 antibody may also be administered with an agent thattargets a member of the TNF and TNFR family of molecules (ligands orreceptors), such as CD40 and CD40L (e.g., human CD40 and human CD40L),OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL/Apo2-L,TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL,TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT,DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDA1, EDA2, TNFR1, Lymphotoxin α/TNFβ,TNFR2, TNFα, LTOR, Lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, andNGFR (see, e.g., Tansey (2009) Drug Discovery Today 00:1).

T cell responses can be stimulated by a combination of anti-CD40antibodies described herein, e.g., 3C3 and 3G5, and one or more of anantagonist (inhibitor or blocking agent) of a protein that inhibits Tcell activation (e.g., immune checkpoint inhibitors), such as CTLA-4,PD-1, PD-L1, PD-L2, and LAG-3, as described above, and any of thefollowing proteins: TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1,TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1,TIM-1, and TIM4-4, and/or one or more of an agonist of a protein thatstimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137),4-1BBL, ICOS, ICOS-L, OX40, OX40L, CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents that modulate one of the above proteins and may becombined with agonist anti-CD40 antibodies, e.g., those describedherein, for treating cancer, include: Yervoy™ (ipilimumab) orTremelimumab (to CTLA-4), galiximab (to B7.1), BMS-936558/nivolumab (toPD-1), MK-3475/pembrolizumab (to PD-1), AMP224 (to B7DC), BMS-936559 (toB7-H1), MPDL3280A/atezolizumab (to B7-H1), MEDI-570 (to ICOS), AMG557(to B7H2), MGA271 (to B7H3), MP321 (to LAG-3), BMS-663513 (to CD137),PF-05082566 (to CD137), CDX-1127 (to CD27), anti-OX40 (Providence HealthServices), huMAbOX40L (to OX40L), Atacicept (to TACI), CP-870893 (toCD40), Lucatumumab (to CD40), Dacetuzumab (to CD40), Muromonab-CD3 (toCD3), Ipilumumab (to CTLA-4).

Other molecules that can be combined with agonist anti-CD40 antibodiesfor the treatment of cancer include antagonists of inhibitory receptorson NK cells or agonists of activating receptors on NK cells. Forexample, anti-CD40 agonist antibodies can be combined with antagonistsof KIR (e.g., lirilumab).

T cell activation is also regulated by soluble cytokines, and anti-CD40antibodies may be administered to a subject, e.g., having cancer, withantagonists of cytokines that inhibit T cell activation or agonists ofcytokines that stimulate T cell activation.

In another embodiment, anti-CD40 antibodies can be used in combinationwith (i) antagonists (or inhibitors or blocking agents) of proteins ofthe IgSF family or B7 family or the TNF family that inhibit T cellactivation or antagonists of cytokines that inhibit T cell activation(e.g., IL-6, IL-10, TGF-β, VEGF; “immunosuppressive cytokines”) and/or(ii) agonists of stimulatory receptors of the IgSF family, B7 family orthe TNF family or of cytokines that stimulate T cell activation, forstimulating an immune response, e.g., for treating proliferativediseases, such as cancer.

Other agents for combination therapies include agents that inhibit ordeplete macrophages or monocytes, including but not limited to CSF-1Rantagonists such as CSF-1R antagonist antibodies including RG7155(WO11/70024, WO11/107553, WO11/131407, WO13/87699, WO13/119716,WO13/132044) or FPA-008 (WO11/140249; WO13169264; WO14/036357).

Anti-CD40 antibodies may also be administered with agents that inhibitTGF-β signaling.

Additional agents that may be combined with an anti-CD40 antibodyinclude agents that enhance tumor antigen presentation, e.g., dendriticcell vaccines, GM-CSF secreting cellular vaccines, CpG oligonucleotides,and imiquimod, or therapies that enhance the immunogenicity of tumorcells (e.g., anthracyclines).

Other therapies that may be combined with an anti-CD40 antibody includetherapies that deplete or block Treg cells, e.g., an agent thatspecifically binds to CD25.

Another therapy that may be combined with an anti-CD40 antibody is atherapy that inhibits a metabolic enzyme such as indoleamine dioxigenase(IDO), dioxigenase, arginase, or nitric oxide synthetase.

Another class of agents that may be used with an anti-CD40 antibodyincludes agents that inhibit the formation of adenosine or inhibit theadenosine A2A receptor.

Other therapies that may be combined with an anti-CD40 antibody fortreating cancer include therapies that reverse/prevent T cell anergy orexhaustion and therapies that trigger an innate immune activation and/orinflammation at a tumor site.

An anti-CD40 antibody may be combined with more than one immuno-oncologyagent, and may be, e.g., combined with a combinatorial approach thattargets multiple elements of the immune pathway, such as one or more ofthe following: a therapy that enhances tumor antigen presentation (e.g.,dendritic cell vaccine, GM-CSF secreting cellular vaccines, CpGoligonucleotides, imiquimod); a therapy that inhibits negative immuneregulation e.g., by inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathwayand/or depleting or blocking Tregs or other immune suppressing cells; atherapy that stimulates positive immune regulation, e.g., with agoniststhat stimulate the CD-137, OX-40, and/or GITR pathway and/or stimulate Tcell effector function; a therapy that increases systemically thefrequency of anti-tumor T cells; a therapy that depletes or inhibitsTregs, such as Tregs in the tumor, e.g., using an antagonist of CD25(e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; a therapythat impacts the function of suppressor myeloid cells in the tumor; atherapy that enhances immunogenicity of tumor cells (e.g.,anthracyclines); adoptive T cell or NK cell transfer includinggenetically modified cells, e.g., cells modified by chimeric antigenreceptors (CAR-T therapy); a therapy that inhibits a metabolic enzymesuch as indoleamine dioxigenase (IDO), dioxigenase, arginase, or nitricoxide synthetase; a therapy that reverses/prevents T cell anergy orexhaustion; a therapy that triggers an innate immune activation and/orinflammation at a tumor site; administration of immune stimulatorycytokines; or blocking of immuno repressive cytokines.

Agonist anti-CD40 antibodies described herein can be used together withone or more of agonistic agents that ligate positive costimulatoryreceptors, blocking agents that attenuate signaling through inhibitoryreceptors, antagonists, and one or more agents that increasesystemically the frequency of anti-tumor T cells, agents that overcomedistinct immune suppressive pathways within the tumor microenvironment(e.g., block inhibitory receptor engagement (e.g., PD-L1/PD-1interactions), deplete or inhibit Tregs (e.g., using an anti-CD25monoclonal antibody (e.g., daclizumab) or by ex vivo anti-CD25 beaddepletion), inhibit metabolic enzymes such as IDO, or reverse/prevent Tcell anergy or exhaustion) and agents that trigger innate immuneactivation and/or inflammation at tumor sites.

Provided herein are methods for stimulating an immune response in asubject comprising administering to the subject an agonist anti-CD40molecule, e.g., an antibody, and one or more additionalimmunostimulatory antibodies, such as an anti-PD-1 antagonist, e.g.,antagonist antibody, an anti-PD-L1 antagonist, e.g., antagonistantibody, an antagonist anti-CTLA-4 antagonist, e.g., antagonistantibody and/or an anti-LAG3 antagonist, e.g., an antagonist antibody,such that an immune response is stimulated in the subject, for exampleto inhibit tumor growth or to stimulate an anti-viral response. In oneembodiment, the additional immunostimulatory antibody (e.g., anantagonist anti-PD-1, an antagonist anti-PD-L1, an antagonistanti-CTLA-4 and/or an antagonist anti-LAG3 antibody) is a humanantibody.

Also provided herein are methods for treating a hyperproliferativedisease (e.g., cancer), comprising administering an agonist anti-CD40antibody and an antagonist PD-1 antibody to a subject. In oneembodiment, the subject is human. In another embodiment, the anti-PD-1antibody is a human sequence monoclonal antibody and the anti-CD40antibody is human sequence monoclonal antibody, such as an antibodycomprising the CDRs or variable regions of 3C3 and 3G5 described hereinor another agonist anti-CD40 antibody described herein.

Suitable PD-1 antagonists for use in the methods described herein,include, without limitation, ligands, antibodies (e.g., monoclonalantibodies and bispecific antibodies), and multivalent agents. In oneembodiment, the PD-1 antagonist is a fusion protein, e.g., an Fc fusionprotein, such as AMP-244. In one embodiment, the PD-1 antagonist is ananti-PD-1 or anti-PD-L1 antibody.

An exemplary anti-PD-1 antibody is nivolumab (BMS-936558) or an antibodythat comprises the CDRs or variable regions of one of antibodies 17D8,2D3, 4H1, 5C4, 7D3, 5F4 and 4A11 described in WO 2006/121168. In certainembodiments, an anti-PD1 antibody is MK-3475 (Lambrolizumab) describedin WO2012/145493; and AMP-514 described in WO 2012/145493. Further knownPD-1 antibodies and other PD-1 inhibitors include those described in WO2009/014708, WO 03/099196, WO 2009/114335, WO 2011/066389, WO2011/161699, WO 2012/145493, U.S. Pat. Nos. 7,635,757 and 8,217,149, andU.S. Patent Publication No. 2009/0317368. Any of the anti-PD-1antibodies disclosed in WO2013/173223 may also be used. An anti-PD-1antibody that competes for binding with, and/or binds to the sameepitope on PD-1 as, as one of these antibodies may also be used incombination treatments. Another approach to target the PD-1 receptor isthe recombinant protein composed of the extracellular domain of PD-L2(B7-DC) fused to the Fc portion of IgG1, called AMP-224.

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering an agonist anti-CD40 antibodyand an antagonist PD-L1 antibody to a subject. In one embodiment, thesubject is human. In another embodiment, the anti-PD-L1 antibody is ahuman sequence monoclonal antibody and the anti-CD40 antibody is humansequence monoclonal antibody, such as an antibody comprising the CDRs orvariable regions of 3C3 and 3G5 described herein or another agonistanti-CD40 antibody described herein.

In one embodiment, the anti-PD-L1 antibody is BMS-936559 (referred to as12A4 in WO 2007/005874 and U.S. Pat. No. 7,943,743), or an antibody thatcomprises the CDRs or variable regions of 3G10, 12A4, 10A5, 5F8, 10H10,1B12, 7H1, 11E6, 12B7 and 13G4, which are described in PCT PublicationWO 07/005874 and U.S. Pat. No. 7,943,743. In certain embodiment ananti-PD-L1 antibody is MEDI4736 (also known as Anti-B7-H1), MPDL3280A(also known as RG7446), MSB0010718C (WO2013/79174), or rHigM12B7. Any ofthe anti-PD-L1 antibodies disclosed in W2013/173223, WO2011/066389,WO2012/145493, U.S. Pat. Nos. 7,635,757 and 8,217,149 and U.S.Publication No. 2009/145493 may also be used. Anti-PD-L1 antibodies thatcompete with and/or bind to the same epitope as that of any of theseantibodies may also be used in combination treatments.

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering an anti-CD40 antibody describedherein and a CTLA-4 antagonist antibody to a subject. In one embodiment,the subject is human. In another embodiment, the anti-CTLA-4 antibody isan antibody selected from the group of. Yervoy™ (ipilimumab or antibody10D1, described in PCT Publication WO 01/14424), tremelimumab (formerlyticilimumab, CP-675,206), monoclonal or an anti-CTLA-4 antibodydescribed in any of the following publications: WO 98/42752; WO00/37504; U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc. Natl.Acad. Sci. USA 95(17):10067-10071; Camacho et al. (2004) J. Cin.Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr etal. (1998) Cancer Res. 58:5301-5304. Any of the anti-CTLA-4 antibodiesdisclosed in WO2013/173223 may also be used.

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering an anti-CD40 antibody and ananti-LAG-3 antibody to a subject. In one embodiment, the subject ishuman. In another embodiment, the anti-PD-L1 antibody is a humansequence monoclonal antibody and the anti-CD40 antibody is humansequence monoclonal antibody, such as an antibody comprising the CDRs orvariable regions of 3C3 or 3G5 described herein or another agonistanti-CD40 antibody described herein. Examples of anti-LAG3 antibodiesinclude antibodies comprising the CDRs or variable regions of antibodies25F7, 26H10, 25E3, 8B7, 11F2 or 17E5, which are described in U.S. PatentPublication No. US2011/0150892, WO10/19570 and WO2014/008218. In oneembodiment, an anti-LAG-3 antibody is BMS-986016. Other art recognizedanti-LAG-3 antibodies that can be used include IMP731 and IMP-321,described in US 2011/007023, WO08/132601, and WO09/44273. Anti-LAG-3antibodies that compete with and/or bind to the same epitope as that ofany of these antibodies may also be used in combination treatments.

Administration of anti-CD40 antibodies described herein and antagonists,e.g., antagonist antibodies, to one or more second target antigens suchas LAG-3 and/or CTLA-4 and/or PD-1 and/or PD-L1 can enhance the immuneresponse to cancerous cells in the patient. Cancers whose growth may beinhibited using the antibodies of the instant disclosure include cancerstypically responsive to immunotherapy and those that are not typicallyresponsive to immunotherapy. Representative examples of cancers fortreatment with the combination therapy of the instant disclosure includethose cancers listed herein.

In certain embodiments, the combination of therapeutic antibodiesdiscussed herein can be administered concurrently as a singlecomposition in a pharmaceutically acceptable carrier, or concurrently asseparate compositions with each antibody in a pharmaceuticallyacceptable carrier. In another embodiment, the combination oftherapeutic antibodies can be administered sequentially. For example, ananti-CTLA-4 antibody and an anti-CD40 antibody can be administeredsequentially, such as anti-CTLA-4 antibody being administered first andanti-CD40 antibody second, or anti-CD40 antibody being administeredfirst and anti-CTLA-4 antibody second. Additionally or alternatively, ananti-PD-1 antibody and an anti-CD40 antibody can be administeredsequentially, such as anti-PD-1 antibody being administered first andanti-CD40 antibody second, or anti-CD40 antibody being administeredfirst and anti-PD-1 antibody second. Additionally or alternatively, ananti-PD-L1 antibody and an anti-CD40 antibody can be administeredsequentially, such as anti-PD-L1 antibody being administered first andanti-CD40 antibody second, or anti-CD40 antibody being administeredfirst and anti-PD-L1 antibody second. Additionally or alternatively, ananti-LAG-3 antibody and an anti-CD40 antibody can be administeredsequentially, such as anti-LAG-3 antibody being administered first andanti-CD40 antibody second, or anti-CD40 antibody being administeredfirst and anti-LAG-3 antibody second.

Furthermore, if more than one dose of the combination therapy isadministered sequentially, the order of the sequential administrationcan be reversed or kept in the same order at each time point ofadministration, sequential administrations can be combined withconcurrent administrations, or any combination thereof. For example, thefirst administration of a combination anti-CTLA-4 antibody and anti-CD40antibody can be concurrent, the second administration can be sequentialwith anti-CTLA-4 antibody first and anti-CD40 antibody second, and thethird administration can be sequential with anti-CD40 antibody first andanti-CTLA-4 antibody second, etc. Additionally or alternatively, thefirst administration of a combination anti-PD-1 antibody and anti-CD40antibody can be concurrent, the second administration can be sequentialwith anti-PD-1 antibody first and anti-CD40 antibody second, and thethird administration can be sequential with anti-CD40 antibody first andanti-PD-1 antibody second, etc. Additionally or alternatively, the firstadministration of a combination anti-PD-L1 antibody and anti-CD40antibody can be concurrent, the second administration can be sequentialwith anti-PD-L1 antibody first and anti-CD40 antibody second, and thethird administration can be sequential with anti-CD40 antibody first andanti-PD-L1 antibody second, etc. Additionally or alternatively, thefirst administration of a combination anti-LAG-3 antibody and anti-CD40antibody can be concurrent, the second administration can be sequentialwith anti-LAG-3 antibody first and anti-CD40 antibody second, and thethird administration can be sequential with anti-CD40 antibody first andanti-LAG-3 antibody second, etc. Another representative dosing schemecan involve a first administration that is sequential with anti-CD40first and anti-CTLA-4 antibody (and/or anti-PD-1 antibody and/oranti-PD-L1 antibody and/or anti-LAG-3 antibody) second, and subsequentadministrations may be concurrent.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-CD40 antibody andan immuno-oncology agent, wherein the immuno-oncology agent is a CD137(4-1BB) agonist, such as an agonistic CD137 antibody. Suitable CD137antibodies include, for example, urelumab or PF-05082566 (WO12/32433).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-CD40 antibody andan immuno-oncology agent, wherein the immuno-oncology agent is an OX40agonist, such as an agonistic OX40 antibody. Suitable OX40 antibodiesinclude, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG7888;WO06/029879).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-CD40 antibody andan immuno-oncology agent, wherein the immuno-oncology agent is a secondCD40 agonist, such as another agonistic CD40 antibody.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-CD40 antibody andan immuno-oncology agent, wherein the immuno-oncology agent is a CD27agonist, such as an agonistic CD27 antibody. Suitable CD27 antibodiesinclude, for example, varlilumab (CDX-1127).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-CD40 antibody andan immuno-oncology agent, wherein the immuno-oncology agent is MGA271(to B7H3) (WO11/109400).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-CD40 antibody andan immuno-oncology agent, wherein the immuno-oncology agent is a KIRantagonist, such as lirilumab.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-CD40 antibody andan immuno-oncology agent, wherein the immuno-oncology agent is an IDOantagonist. Suitable IDO antagonists include, for example, INCB-024360(WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximod, NLG-919(WO09/73620, WO09/1156652, WO11/56652, WO12/142237) or F001287.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-CD40 antibody andan immuno-oncology agent, wherein the immuno-oncology agent is aToll-like receptor agonist, e.g., a TLR2/4 agonist (e.g., BacillusCalmette-Guerin); a TLR7 agonist (e.g., Hiltonol or Imiquimod); a TLR7/8agonist (e.g., Resiquimod); or a TLR9 agonist (e.g., CpG7909).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-CD40 antibody andan immuno-oncology agent, wherein, the immuno-oncology agent is a TGF-βinhibitor, e.g., GC1008, LY2157299, TEW7197, or IMC-TR1.

In one aspect, an anti-CD40 antibody is sequentially administered priorto administration of a second agent, e.g., an immuno-oncology agent. Inone aspect, an anti-CD40 antibody is administered concurrently with thesecond agent, e.g., an immunology-oncology agent. In yet one aspect, ananti-CD40 antibody is sequentially administered after administration ofthe second agent. The administration of the two agents may start attimes that are, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days,7 days, or one or more weeks apart, or administration of the secondagent may start, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes,3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5days, 7 days, or one or more weeks after the first agent has beenadministered.

In certain aspects, an anti-CD40 antibody and a second agent, e.g., animmuno-oncology agent, are administered simultaneously, e.g., areinfused simultaneously, e.g., over a period of 30 or 60 minutes, to apatient. Alternatively, the anti-CD40 antibody may be co-formulated witha second agent, e.g., an immuno-oncology agent.

Optionally, the anti-CD40 is administered as the sole immunotherapeuticagent, or a combination of the anti-CD40 antibody and one or moreadditional immunotherapeutic antibodies (e.g., anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibody) can be furthercombined with an immunogenic agent, such as cancerous cells, purifiedtumor antigens (including recombinant proteins, peptides, andcarbohydrate molecules), cells, and cells transfected with genesencoding immune stimulating cytokines (He et al. (2004) J. Immunol.173:4919-28). Non-limiting examples of tumor vaccines that can be usedinclude peptides of melanoma antigens, such as peptides of gpl00, MAGEantigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected toexpress the cytokine GM-CSF (discussed further below). The anti-CD40antibody and one or more additional antibodies (e.g., anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies) can also befurther combined with standard cancer treatments. For example, theanti-CD40 antibody and one or more additional antibodies (e.g.,anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3antibodies) can be effectively combined with chemotherapeutic regimes.In these instances, it is possible to reduce the dose of otherchemotherapeutic reagent administered with the combination of theinstant disclosure (Mokyr et al. (1998) Cancer Research 58: 5301-5304).An example of such a combination is a combination of anti-CD40 agonistantibody (with or without and an additional antibody, such asanti-CTLA-4 antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1antibodies and/or anti-LAG-3 antibodies) in combination with decarbazinefor the treatment of melanoma. Another example is a combination ofanti-CD40 antibody (with or without anti-CTLA-4 antibodies and/oranti-PD-1 antibodies and/or anti-PD-L1 antibodies and/or LAG-3antibodies) in combination with interleukin-2 (IL-2) for the treatmentof melanoma. The scientific rationale behind the combined use of ananti-CD40 antibody and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1and/or anti-LAG-3 antibodies with chemotherapy is that cell death, whichis a consequence of the cytotoxic action of most chemotherapeuticcompounds, should result in increased levels of tumor antigen in theantigen presentation pathway. Other combination therapies that mayresult in synergy with an anti-CD40 antibody (with or without ananti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3antibody) include radiation, surgery, or hormone deprivation. Each ofthese protocols creates a source of tumor antigen in the host.Angiogenesis inhibitors can also be combined with a combined ananti-CD40 antibody and an anti-CTLA-4 antibody and/or anti-PD-1 antibodyand/or anti-PD-L1 antibody and/or anti-LAG-3 antibody. Inhibition ofangiogenesis leads to tumor cell death, which can be a source of tumorantigen fed into host antigen presentation pathways.

An anti-CD40 agonist antibody as sole immunotherapeutic agent, or acombination of CD40 agonistic and CTLA-4 and/or PD-1 and/or PD-L1 and/orLAG-3 blocking antibodies also can be used in combination withbispecific antibodies that target Fca or Fcγ receptor-expressingeffector cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. The T cell arm of these responses would be augmented by theuse of a combined anti-CD40 antibody and anti-CTLA-4 antibody and/oranti-PD-1 antibody and/or anti-PD-L1 antibody and/or anti-LAG-3antibody.

In another example, an anti-CD40 agonist antibody as the soleimmunotherapeutic agent or a combination of an anti-CD40 antibody andadditional immunostimulating agent, e.g., anti-CTLA-4 antibody and/oranti-PD-1 antibody and/or anti-PD-L1 antibody and/or LAG-3 agent, e.g.,antibody, can be used in conjunction with an anti-neoplastic antibody,such as Rituxan® (rituximab), Herceptin® (trastuzumab), Bexxar®(tositumomab), Zevalin® (ibritumomab), Campath® (alemtuzumab),Lymphocide® (eprtuzumab), Avastin® (bevacizumab), and Tarceva®(erlotinib), and the like. By way of example and not wishing to be boundby theory, treatment with an anti-cancer antibody or an anti-cancerantibody conjugated to a toxin can lead to cancer cell death (e.g.,tumor cells) which would potentiate an immune response mediated by theimmunostimulating agent, e.g., CD40, CTLA-4, PD-1, PD-L1 or LAG-3 agent,e.g., antibody. In an exemplary embodiment, a treatment of ahyperproliferative disease (e.g., a cancer tumor) can include ananti-cancer agent, e.g., antibody, in combination with anti-CD40 andoptionally an additional immunostimulating agent, e.g., anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent, e.g.,antibody, concurrently or sequentially or any combination thereof, whichcan potentiate an anti-tumor immune responses by the host.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofproteins, which are expressed by the tumors and which areimmunosuppressive. These include, among others, TGF-β (Kehrl et al.(1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992)Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996)Science 274: 1363-1365). Antibodies to each of these entities can befurther combined with an anti-CD40 antibody with or without anadditional immunostimulating agent, e.g., an anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent, such as antibody,to counteract the effects of immunosuppressive agents and favoranti-tumor immune responses by the host.

Other agents, e.g., antibodies, that can be used to activate host immuneresponsiveness can be further used in combination with an anti-CD40antibody with or without an additional immunostimulating agent, such asanti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3antibody. These include molecules on the surface of dendritic cells thatactivate DC function and antigen presentation. Anti-CD40 antibodies(Ridge et al., supra) can be used in conjunction with an anti-CD40antibody and optionally an additional immunostimulating agent, e.g., ananti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent,e.g., antibody. Other activating antibodies to T cell costimulatorymolecules Weinberg et al., supra, Melero et al. supra, Hutloff et al.,supra, may also provide for increased levels of T cell activation.

As discussed above, bone marrow transplantation is currently being usedto treat a variety of tumors of hematopoietic origin. Anti-CD40immunotherapy alone or combined with an anti-CTLA-4 antibody and/oranti-PD-1 antibody and/or anti-PD-L1 antibody and/or anti-LAG-3 antibodycan be used to increase the effectiveness of the donor engrafted tumorspecific T cells.

Several experimental treatment protocols involve ex vivo activation andexpansion of antigen specific T cells and adoptive transfer of thesecells into recipients in order to antigen-specific T cells against tumor(Greenberg & Riddell, supra). These methods can also be used to activateT cell responses to infectious agents such as CMV. Ex vivo activation inthe presence of anti-CD40 with or without an additionalimmunostimulating therapy, e.g., anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 and/or anti-LAG-3 antibodies can be expected to increase thefrequency and activity of the adoptively transferred T cells.

Provided herein are methods for altering an adverse event associatedwith treatment of a hyperproliferative disease (e.g., cancer) with animmunostimulatory agent, comprising administering an anti-CD40 antibodywith or without anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/oranti-LAG-3 agent, e.g., antibody, to a subject. For example, the methodsdescribed herein provide for a method of reducing the incidence ofimmunostimulatory therapeutic antibody-induced colitis or diarrhea byadministering a non-absorbable steroid to the patient. As used herein, a“non-absorbable steroid” is a glucocorticoid that exhibits extensivefirst pass metabolism such that, following metabolism in the liver, thebioavailability of the steroid is low, i.e., less than about 20%. In oneembodiment described herein, the non-absorbable steroid is budesonide.Budesonide is a locally-acting glucocorticosteroid, which is extensivelymetabolized, primarily by the liver, following oral administration.ENTOCORT EC® (Astra-Zeneca) is a pH- and time-dependent oral formulationof budesonide developed to optimize drug delivery to the ileum andthroughout the colon. ENTOCORT EC® is approved in the U.S. for thetreatment of mild to moderate Crohn's disease involving the ileum and/orascending colon. The usual oral dosage of ENTOCORT EC® for the treatmentof Crohn's disease is 6 to 9 mg/day. ENTOCORT EC® is released in theintestines before being absorbed and retained in the gut mucosa. Once itpasses through the gut mucosa target tissue, ENTOCORT EC® is extensivelymetabolized by the cytochrome P450 system in the liver to metaboliteswith negligible glucocorticoid activity. Therefore, the bioavailabilityis low (about 10%). The low bioavailability of budesonide results in animproved therapeutic ratio compared to other glucocorticoids with lessextensive first-pass metabolism. Budesonide results in fewer adverseeffects, including less hypothalamic-pituitary suppression, thansystemically-acting corticosteroids. However, chronic administration ofENTOCORT EC® can result in systemic glucocorticoid effects such ashypercorticism and adrenal suppression. See PDR 58^(th) ed. 2004;608-610.

In still further embodiments, the anti-CD40 antibody, with or withoutimmunostimulatory therapeutic antibodies anti-CD40 and optionallyanti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3antibodies, in conjunction with a non-absorbable steroid can be furthercombined with a salicylate. Salicylates include 5-ASA agents such as,for example: sulfasalazine (AZULFIDINE®, Pharmacia & UpJohn); olsalazine(DIPENTUM®, Pharmacia & UpJohn); balsalazide (COLAZAL®, SalixPharmaceuticals, Inc.); and mesalamine (ASACOL®, Procter & GamblePharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.;ROWASA®, Solvay).

In accordance with the methods described herein, a salicylate isadministered in combination with anti-CD40, with or without anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 and/or LAG-3 antibodies, and anon-absorbable steroid for the purpose of decreasing the incidence ofcolitis induced by the immunostimulatory antibodies. Thus, for example,methods for reducing the incidence of colitis induced by theimmunostimulatory antibodies described herein encompass administering asalicylate and a non-absorbable concurrently or sequentially (e.g., asalicylate is administered 6 hours after a non-absorbable steroid), orany combination thereof. Further, a salicylate and a non-absorbablesteroid can be administered by the same route (e.g., both areadministered orally) or by different routes (e.g., a salicylate isadministered orally and a non-absorbable steroid is administeredrectally), which may differ from the route(s) used to administer theanti-CD40 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/oranti-LAG-3 antibodies.

The anti-CD40 antibodies and combination antibody therapies describedherein may also be used in conjunction with other well known therapiesthat are selected for their particular usefulness against the indicationbeing treated (e.g., cancer). Combinations of the anti-CD40 antibodiesdescribed herein may be used sequentially with known pharmaceuticallyacceptable agent(s).

For example, the anti-CD40 antibodies and combination antibody therapiesdescribed herein can be used in combination (e.g., simultaneously orseparately) with an additional treatment, such as irradiation,chemotherapy (e.g., using camptothecin (CPT-11), 5-fluorouracil (5-FU),cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin,paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-fu, orcamptothecin+apo2/TRAIL (a 6× combo)), one or more proteasome inhibitors(e.g., bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH3I-2′(bcl-xl inhibitor), indoleamine dioxygenase-1 inhibitor (e.g.,INCB24360, indoximod, NLG-919, or F001287), AT-101 (R-(−)-gossypolderivative), ABT-263 (small molecule), GX-15-070 (obatoclax), or MCL-1(myeloid leukemia cell differentiation protein-1) antagonists), iAP(inhibitor of apoptosis protein) antagonists (e.g., smac7, smac4, smallmolecule smac mimetic, synthetic smac peptides (see Fulda et al., NatMed 2002; 8:808-15), ISIS23722 (LY2181308), or AEG-35156 (GEM-640)),HDAC (histone deacetylase) inhibitors, anti-CD20 antibodies (e.g.,rituximab), angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenicagents targeting VEGF and VEGFR (e.g., Avastin), synthetic triterpenoids(see Hyer et al., Cancer Research 2005; 65:4799-808), c-FLIP (cellularFLICE-inhibitory protein) modulators (e.g., natural and syntheticligands of PPARγ (peroxisome proliferator-activated receptor γ), 5809354or 5569100), kinase inhibitors (e.g., Sorafenib), Trastuzumab,Cetuximab, Temsirolimus, mTOR inhibitors such as rapamycin andtemsirolimus, Bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKTinhibitors, Lenalildomide, GSK3β inhibitors, IAP inhibitors and/orgenotoxic drugs.

The anti-CD40 antibodies and combination antibody therapies describedherein can further be used in combination with one or moreanti-proliferative cytotoxic agents. Classes of compounds that may beused as anti-proliferative cytotoxic agents include, but are not limitedto, the following:

Alkylating agents (including, without limitation, nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes):Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN™) fosfamide,Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, and Temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists,pyrimidine analogs, purine analogs and adenosine deaminase inhibitors):Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.

Suitable anti-proliferative agents for combining with agonist anti-CD40antibodies, without limitation, taxanes, paclitaxel (paclitaxel iscommercially available as TAXOL™), docetaxel, discodermolide (DDM),dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothiloneB, epothilone C, epothilone D, epothilone E, epothilone F,furanoepothilone D, desoxyepothilone B1, [17]-dehydrodesoxyepothilone B,[18]dehydrodesoxyepothilones B, C12,13-cyclopropyl-epothilone A, C6-C8bridged epothilone A, trans-9,10-dehydroepothilone D,cis-9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone B10,discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO,ABJ-789, XAA296A (Discodermolide), TZT-1027 (soblidotin), ILX-651(tasidotin hydrochloride), Halichondrin B, Eribulin mesylate (E-7389),Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703, Maytansinoidimmunoconjugates (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992(ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin,17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol,cyclostreptin, isolaulimalide, laulimalide,4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, andcryptothilone 1, in addition to other microtubuline stabilizing agentsknown in the art.

In cases where it is desirable to render aberrantly proliferative cellsquiescent in conjunction with or prior to treatment with anti-CD40antibodies described herein, hormones and steroids (including syntheticanalogs), such as 17a-Ethinylestradiol, Diethylstilbestrol,Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate,Testolactone, Megestrolacetate, Methylprednisolone, Methyl-testosterone,Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone,Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide,Flutamide, Toremifene, ZOLADEXM, can also be administered to thepatient. When employing the methods or compositions described herein,other agents used in the modulation of tumor growth or metastasis in aclinical setting, such as antimimetics, can also be administered asdesired.

Methods for the safe and effective administration of chemotherapeuticagents are known to those skilled in the art. In addition, theiradministration is described in the standard literature. For example, theadministration of many of the chemotherapeutic agents is described inthe Physicians' Desk Reference (PDR), e.g., 1996 edition (MedicalEconomics Company, Montvale, N.J. 07645-1742, USA); the disclosure ofwhich is incorporated herein by reference thereto.

The chemotherapeutic agent(s) and/or radiation therapy can beadministered according to therapeutic protocols well known in the art.It will be apparent to those skilled in the art that the administrationof the chemotherapeutic agent(s) and/or radiation therapy can be varieddepending on the disease being treated and the known effects of thechemotherapeutic agent(s) and/or radiation therapy on that disease.Also, in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents on the patient, and in view of the observed responsesof the disease to the administered therapeutic agents.

VI. Outcomes

As shown in the Examples herein, co-administration of an anti-CD40antibody with one or more additional therapeutic agents (e.g., solubleCD40 ligand or another antibody, such as an anti-PD-1 antibody, ananti-PD-L1 antibody, an anti-CTLA-4 antibody, and/or an anti-LAG-3antibody) provides improved efficacy compared to treatment with theantibody alone or with the one or more additional therapeutic agents inthe absence of antibody therapy. Preferably, a combination of ananti-CD40 antibody with one or more additional therapeutic agentsexhibits therapeutic synergy.

“Therapeutic synergy” refers to a phenomenon where treatment of patientswith a combination of therapeutic agents manifests a therapeuticallysuperior outcome to the outcome achieved by each individual constituentof the combination used at its optimum dose (T. H. Corbett et al., 1982,Cancer Treatment Reports, 66, 1187). In this context a therapeuticallysuperior outcome is one in which the patients either a) exhibit fewerincidences of adverse events while receiving a therapeutic benefit thatis equal to or greater than that where individual constituents of thecombination are each administered as monotherapy at the same dose as inthe combination, or b) do not exhibit dose-limiting toxicities whilereceiving a therapeutic benefit that is greater than that of treatmentwith each individual constituent of the combination when eachconstituent is administered in at the same doses in the combination(s)as is administered as individual components. In xenograft models, acombination, used at its maximum tolerated dose, in which each of theconstituents will be present at a dose generally not exceeding itsindividual maximum tolerated dose, manifests therapeutic synergy when,for example, a decrease in tumor growth is achieved by administration ofthe combination which is greater than the value of the decrease in tumorgrowth of the best constituent when the constituent is administeredalone.

Thus, in combination, the components of such combinations have anadditive or superadditive effect on suppressing tumor growth, ascompared to monotherapy with the anti-CD40 antibody or treatment withthe additional therapeutic agent(s) in the absence of antibody therapy.By “additive” is meant a result that is greater in extent (e.g., in thedegree of reduction of tumor mitotic index or of tumor growth or in thedegree of tumor shrinkage or the frequency and/or duration ofsymptom-free or symptom-reduced periods) than the best separate resultachieved by monotherapy with each individual component, while“superadditive” is used to indicate a result that exceeds in extent thesum of such separate results. In one embodiment, the additive effect ismeasured as slowing or stopping of tumor growth. The additive effect canalso be measured as, e.g., reduction in size of a tumor, reduction oftumor mitotic index, reduction in number of metastatic lesions overtime, increase in overall response rate, or increase in median oroverall survival. In another embodiment, the additive effect is measuredas increasing induction of CD95 expression when incubated with Ramoscells, increasing B cell proliferation when incubated with human Bcells, and/or increasing increased induction of IL12p40 expression whenincubated with dendritic cells.

One non-limiting example of a measure by which effectiveness of atherapeutic treatment can be quantified is by calculating the log 10cell kill, which is determined according to the following equation:log 10 cell kill=TC(days)/3.32×Tdin which T C represents the delay in growth of the cells, which is theaverage time, in days, for the tumors of the treated group (T) and thetumors of the control group (C) to have reached a predetermined value (1g, or 10 mL, for example), and Td represents the time, in days necessaryfor the volume of the tumor to double in the control animals. Whenapplying this measure, a product is considered to be active if log 10cell kill is greater than or equal to 0.7 and a product is considered tobe very active if log 10 cell kill is greater than 2.8. Using thismeasure, a combination, used at its own maximum tolerated dose, in whicheach of the constituents is present at a dose generally less than orequal to its maximum tolerated dose, exhibits therapeutic synergy whenthe log 10 cell kill is greater than the value of the log 10 cell killof the best constituent when it is administered alone. In an exemplarycase, the log 10 cell kill of the combination exceeds the value of thelog 10 cell kill of the best constituent of the combination by at least0.1 log cell kill, at least 0.5 log cell kill, or at least 1.0 log cellkill.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES Example 1

Generation of CD40-Specific Human Monoclonal Antibodies

Human anti-CD40 monoclonal antibodies were generated by immunizing theH2L2 strain of Harbour® transgenic mice with a soluble human CD40antigen. Harbour® transgenic mice have had the endogenous mouse heavychain (HC) and kappa light chain (Y-chain) DNA sequences knocked out andhave had sequences for the human variable (V) regions and rat constant(C) regions stably incorporated into the mouse genome.

Antigen and Immunization: The antigen was a soluble fusion proteincomprising a CD40 extracellular domain fused with an antibody Fc domain(R&D Systems®), or a recombinant human CD40-msG2a chimeric protein (madein-house). The antigen was mixed with Complete Freund's (Sigma) adjuvantfor the first immunization. Thereafter, the antigen was mixed withIncomplete Freund's (Sigma). Additional mice were immunized with thesoluble CD40 protein in MPL plus TDM adjuvant system (Sigma®).5-micrograms soluble recombinant CD40 antigen in PBS or 5×10⁶ NSO cellstransfected for surface expression of human CD40 in PBS were mixed 1:1with the adjuvant. Mice were injected with 200 microliters of theprepared antigen into the peritoneal cavity every 14 days. Animals thatdeveloped anti-CD40 titers were given an iv injection of 5-10 microgramssoluble recombinant CD40 antigen three to four days prior to fusion.Mouse spleens were harvested, and the isolated splenocytes used forhybridoma preparation.

Hybridoma Preparation: The P3×63Ag8.653 murine myeloma cell line (ATCCCRL 1580) was used for the fusions. RPMI 1640 (Invitrogen®) containing10% FBS was used to culture the myeloma cells. Additional mediasupplements were added to the Hybridoma growth media, which included: upto 10% Hybridoma Enhancing Supplement (Sigma), 10% FBS (Sigma),L-glutamine (Gibco®) 0.1% gentamycin (Gibco), 2-mercaptoethanol (Gibco),with HAT (Sigma; 1.0×10⁴ M hypoxanthine, 4.0×10⁻⁷ M aminopterin,1.6×10⁻⁵ M thymidine media.

Spleen cells were mixed with the P3×63Ag8.653myeloma cells in a 6:1ratio and pelleted by centrifugation. Polyethylene glycol was addeddropwise with careful mixing to facilitate fusion. Hybridomas wereallowed to grow out for one to two weeks until visible colonies becomeestablished. Supernatant was harvested and used for initial screeningfor rat IgG via ELISA using a human soluble CD40 fusion protein and arat Fc specific detection. IgG positive supernatants were then assayedfor CD40 specificity via flow cytometry. The hybridomas were alsoscreened for cross-reactivity with cynomolgus macaque CD40 and all werepositive for binding.

Hybridoma cells were expanded and cell pellets were frozen for RNAisolation and sequencing. The V_(H) and V_(L) coding regions of humanmAbs were identified using RNA from the corresponding hybridomas. RNAwas reverse transcribed to cDNA, the V coding regions were amplified byPCR and the PCR product was sequenced, inserted into human IgG2 vector,transiently expressed and purified by protein A column chromatographywhich led to the isolation of a number of antibodies of particularinterest, which were designated as 3C3, 3G5, 1B4, 3B6, 6H6, 6H6, 2E1.2,1B5-NK (in the latter case following N75K modification on FR3 of theheavy chain), and 3B6-NS (following N63S modification of antibody 3B6 onFR3 of the light chain to remove an N-linked glycosylation site).

Tables 1, 2, and 3 summarize the germline information and amino acidsequences of the V_(H) and V_(L) regions of the human mAbs (in the caseof the amino acid sequences, the Complementarity Determining Regions(CDRs) are underlined). The corresponding nucleic acid sequences areprovided in the sequence table headed “Summary of Sequence Listing” atthe end of these Examples.

TABLE 1 Germline Data Germline mAb VH/VL V D J 3G5 H IGHV3-33*01 FIGHD3-10*01 F IGHJ4*02 F (VH3-33) (D3-10) (JH4b) L IGKV3-15*01 FIGKJ5*01 F (L2) (JK5) 3C3 H IGHV3-33*01 F IGHD3-10*02 F IGHJ4*02 F(VH3-33) (D4-b) (JH4b) L IGKV1-27*01 F IGKJ3*01 F (A20) (JK3) 3B6 HIGHV3-23*01 F IGHD2-15*01 F IGHJ6*02 F (VH3-23) (D2-15) (JH6b) LIGKV2-28*01 F IGKJ1*01 F (A19) (JK1) 6H6 H IGHV3-33*01 F IGHD3-10*01 FIGHJ4*02 F (VH3-33) (D3-10) (JH4b) L IGKV3-15*01 F IGKJ4*01 F (L2) (JK4)1B4 H IGHV3-23*01 F IGHD1-26*01 F IGHJ6*02 F (VH3-23) (D2-15) (JH6b) LIGKV2-28*01 F IGKJ1*01 F (A19) (JK1) 1B5- H IGHV3-33*03 F IGHD6-19*01 FIGHJ2*01 F NK (VH3-33) (D2-15) (JH2) L IGKV1-27*01 F IGKJ2*01 F (A20)(JK2) 2 H IGHV3-33*01 F IGHD3-10*01 F IGHJ4*02 F E1.2 (VH3-33) (D3-10)(JH4B) L2 IGKV3-15*01 F IGKJ4*01 F (L2) (JK4) 3B6- H IGHV3-23*01 FIGHD2-15*01 F IGHJ6*02 F NS (VH3-23) (D2-15) (JH6b) L2 IGKV2-28*01 FIGKJ1*01 F (A19) (JK1)

TABLE 2 CDR Sequences mAb VH/VL CDR1 SEQ ID NO CDR2 SEQ ID NO CDR3SEQ ID NO 3G5 H SNGIH 5, 6 VIWSDGSNKFYADSVKG 7, 8 ASGSGSYYNFFDY 9, 10(GFTFSSN) (WSDGSN) (ASGSGSYYNFFDY) L RASQSVRSNLA 11, 12 GASTRAT 13, 14QQHNKWIT 15, 16 (RASQSVRSNLA) (GASTRAT) (QQHNKWIT) 3C3 H RYGMY 19, 20VIWYDGSYKYYADSVKG 21, 22 ESPWYYFDY 23, 24 (GFIFSRY) (WYDGSY) (ESPWYYFDY)L RASQGISNYLA 25, 26 AASTLQS 27, 28 QKYKSAPFT 29, 30 (RASQGISNYLA)(AASTLQS) (QKYKSAPFT) 3B6 H SYAMS 33, 34 GITGTGGSTYYADSVKG 35, 36RAGGSFYYYYGMDV 37, 38 (GFTFSSY) (TGTGGS) (RAGGSFYYYYGMDV) LRSSQSLLHSTGYNYLD 39, 40 LGSNRAS 41, 42 MQALQTPWT 43, 44(RSSQSLLHSTGYNYLD) (LGSNRAS) (MQALQTPWT) 6H6 H SYGMH 47, 48VIWDDGSNKYYADSVKG 49, 50 AGGSGRYYNYFDY 51, 52 (GFTLSSY) (WDDGSN)(AGGSGRYYNYFDY) L RASQSVRSNLA 53, 54 GASTRAT 55, 56 QQHNNWLT 57, 58(RASQ1SVRSNLA) (GASTRAT) (QQHNNWLT) 1B4 H SYAMT 61, 62 GITGSGANTFYTDSVKG63, 64 RNGGSYYYYYGMDV 65, 66 (GFTFSSY) (TGSGAN) (RNGGSYYYYYGMDV) LRSSQSLLHSSGYNYLD 67, 68 LGSNRAS 69, 70 MQALQIPWT 71, 72(RSSQSLLHSSGYNYLD) (LGSNRAS) (MQALQIPWT) 1B5-NK H SFGMH 103, 104LIWFDGSSKYYADSVKG 105, 106 GFAAVAGWYFDF 107, 108 (GFTFSSF) (WFDGSS)(GFAAVAGWYFDF) L RASQGVRKYLA 109, 110 AASTLQS 111, 112 QKYFSAPYT113, 114 (RASQSVRSNLA) (AASTLQS) (QKYFSAPYT) 2E1.2 H SYGMH 89, 90VIWDDGSNKYYADSVKG 91, 92 AGSSGRYYNYFDY 93, 94 (GFTFSSY) (WDDGSN)(AGSSGRYYNYFDY) L RASQSVRSNLA 95, 96 GASTRAT 97, 98 QQYNKWLI 99, 100(RASQSVRSNLA) (GASTRAT) (QQYNKWLI) 3B6-NS H SYAMS 75, 76GITGTGGSTYYADSVKG 77, 78 RAGGSFYYYYGMDV 79, 80 (GFTFSSY) (TGTGGS)(RAGGSFYYYYGMDV) L RSSQSLLHSTGYNYLD 81, 82 LGSNRAS 83, 84 MQALQTPWT85, 86 (RSSQSLLHSTGYNYLD) (LGSNRAS) (MQALQTPWT)

TABLE 3 Full-Length Variable Region Sequences mAb VH/VL SEQ ID NOSequence 3G5 H 3 QVQLVESGGGVVQPGKSLRLSCAASGFTFS SNGIH WVRQAPGKGLEWVAVIWSDGS NKFYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR ASGSGSYYNFFDY WGQGTLVTVSS L 4 EIVMTQSPATLSVSPGERATLSC RASQSVRSNLA WYQQKPGQAPRLLIYGASTRAT GIPARFSGSGSGTEFTLTINSLQSEDFAVYYC QQHNKWIT FGQGTRLEIK 3C3 H 17QVQLVESGGGVVQPGRSLRLSCAGSGFIFS RYGMY WVRQAPGKGLEWVA VIWYDGS YKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR ESPWYYFDY WGQGT LVTVSS L 18DIQMTQSPSSLSASVGDRVTITC RASQGISNYLA WYQQKPGKVPKLLIY AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYKSAPFTFGPGTKVDIK 3B6 H 31EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS GITGTGG STYYADSVKGRFTISRDNSKNTLYVQMNSLRAEDTAVYYCAK RAGGSFYYYYGMDV WGQGTTVTVSS L 32DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSTGYNYLD WYLQKPGQSPQLLIY LG SNRASGVPDRFNGSGSGTDFTLKISRVEAEDFGVYYC MQALQTPWT FGHGTKVEIK 6H6 H 45QVQLVESGGGVVQPGRSLRFSCAASGFTLS SYGMH WVRQAPGKGLEWVA VIWDDGS NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR AGGSGRYYNYFD YW GQGTLVTVSS L 46EIVMTQSPATLSVSPGERATLSC RASQSVRSNLA WYQQKPGQAPRLLIY GASTRATGIPARFSGSGSGTDFTLTISSLQSEDFAVYYC QQHNNWLT FGGGTKVEIK 1B4 H 59EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMT WVRQVPGKGLEWVS GITGSGA NTFYTDSVKGRFTISRDNSNNSLYLQMNSLRADDTAVYYCAK RNGGSYYYYYGMDV WGQGTTVTVSS L 60DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSSGYNYLD WYLQKPGQSPQLLIY LG SNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALQIPWT FGQGTKVEIK 1B5-NK H 101QVQLVESGGGVVQPGRSLRLSCAASGFTFS SFGMH WVRQAPGKGLEWV TLIWFDGS SKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVR GFAAVAGWYFDF WG RGTLVTVSS L 102DIQMTQSPSSLSASVGDRVTITC RASQGVRKYLA WYQQKPGKVPKLLIY AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QKYFSAPYT FGQGTKLEIK 2E1.2 H 87QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA VIWDDGS NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARA GSSGRYYNYFDY W GQGTLVTVSS L 88EIVMTQSPATLSVSPGERATLSC RASQSVRSNLA WYQQKPGQAPRLLIY GASTRATGIPDRFSGSGSGTEFTLTISSLQSEDFAVYHC QQYNKWLI FGGGTKVEIK 3B6-NS H 73EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS GITGTGG STYYADSVKGRFTISRDNSKNTLYVQMNSLRAEDTAVYYCAK RAGGSFYYYYGMDV WGQGTTVTVSS L 74DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSTGYNYLD WYLQKPGQSPQLLIY LG SNRASGVPDRFSGSGSGTDFTLKISRVEAEDFGVYYC MQALQTPWT FGHGTKVEIKThe full amino acid sequences of the heavy and light chains of theantibody 3C3 was as follows:

Light chain sequence with leader sequence removed) (SEQ ID NO: 136)DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYKSAPFTFGPGT KVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGECHeavy chain sequence (with leader sequence removed) (SEQ ID NO: 135)QVQLVESGGGVVQPGRSLRLSCAGSGFIFSRYGMYWVRQAPGKGLEWVAVIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARESPW YYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In each case the variable sequence is shown in italics and the constantdomain is shown in bold. The constant domain sequence is an IgG2sequence from which the C-terminal lysines have been removed.

The same constant domain sequence was used for the other antibodies withtheir respective variable sequences as listed above.

Example 2

Determination of Affinity and Rate Constants of Human mAbs by Bio-LayerInterferometry (BLI)

Binding affinity and binding kinetics of various human anti-CD40antibodies were examined by bio-layer interferometry (BLI) using anOctet™ QK^(e) instrument (Pall ForteBio®, Menlo Park, Calif.) accordingto the manufacturer's guidelines.

Purified antibodies from Example 1 were captured on Anti-Human FcCapture (AHC) biosensors (Fortebio Product No. 18-5060). Each antibodywas prepared in dilution buffer (10 mMPO4+150 mM NaCl+1 mg/mL BSA+0.5%Tween 20, pH 7.2) to 0.5 μg/mL and loaded on freshly hydrated AHCbiosensors for 35-50 sec at 25° C. and 1000 rpm plate shake speed toachieve a target response of 0.2 nm. Low levels of ligand were capturedto limit any effects of mass transport of analyte on kinetic parameters.For one assay, eight biosensors were loaded with the same antibody.

Binding was determined by exposing six of the antibody loaded biosensorsto analyte: soluble human CD40-MsIgG2a (Celldex®, 60 kD by SDS-PAGE).Affinity measurements were determined using 2-fold serial dilutions ofanalyte ranging from 3.13 to 0.098 nM in dilution buffer at 25° C. and1000 rpm plate shake speed. Association of the antibody loadedbiosensors in analyte wells was carried out for 1200 seconds, thebiosensors were then moved to dilution buffer wells for 2.5 hrs (9000sec) for dissociation measurements.

Corresponding controls were conducted in each case by keeping the tworemaining biosensors with captured antibody in dilution buffer wells forassociation and dissociation steps. The data for the control biosensorswas used to subtract background and account for biosensor drift andantibody dissociation from the biosensors.

Fortebio's Data Analysis Software version 8.2.0.7 (Pall ForteBio, MenloPark, Calif.) was used in each case to derive kinetic parameters fromthe concentration series of analyte in dilution buffer binding tocaptured antibody. The association and dissociation curves were fittedto a 1:1 binding model using the data analysis software according to themanufacturer's guidelines.

The affinity and kinetic parameters (with background subtracted) asdetermined are shown in FIG. 1, where kon=rate constant of association,kdis=rate constant of dissociation, and K_(D)=dissociation equilibriumbinding constant, determined by the ratio kdis/kon.

Example 3

Assays to Determine Human mAb Binding Characteristics to CD40

Microtiter plates were coated with recombinant human CD40-Fc in PBS, andthen blocked with 5% bovine serum albumin in PBS. Protein A purifiedhuman mAbs from Example 1 and an isotype control were added at variousconcentrations and incubated at 37° C. The plates were washed withPBS/Tween and then incubated with a goat-anti-human IgG F(ab′)2-specificpolyclonal reagent conjugated to horseradish peroxidase at 37° C. Afterwashing, the plates were developed with HRP substrate, and analyzed atOD 450-650 using a microtiter plate reader. Representatives bindingcurves are shown in FIG. 2.

To establish that cynomolgus macques are a relevant model for testinganti-CD40 mAbs, purified macaque PBMC's or human PBMC's were incubatedwith varying concentrations of anti-human CD40 mAb for 20 minutes atroom temperature on a plate shaker. The cells were then washed twicewith PBS containing 0.1% BSA and 0.05% NaN₃ (PBA). A goat anti-human IgGFc-PE antibody was added for 20 minutes at room temperature on a plateshaker. B cells were identified by subsequent staining with anallophycocyanin (APC) conjugated CD20 antibody. Cells were analyzed byflow cytometry and binding curves are shown in FIG. 3, which indicatesimilar binding to CD40 from macaque and human.

Example 4

Blocking of sCD40L Binding by ELISA

The effect of the human mAbs from Example 1 on the binding of solubleCD40 Ligand (sCD40L) to CD40 protein was measured by ELISA. A microtiterplate was coated with 2 μg/ml soluble recombinant human CD40/Fc chimerafrom R&D Systems, then blocked with 5% PBA. The anti-CD40 antibodies([final]=100 μg/mL) were added to the plate, followed by soluble humanrecombinant CD40L-biotin from Immunex ([final]=0.5 μg/mL). CD40-capturedrCD40L was detected with streptavidin-IRP and substrate Super Blue TMB.The results are shown in FIGS. 4A and B with controls as indicated.

Example 5

Binding to CD40 Cells

The ability of anti-CD40 human mAbs to bind to CD40 on cells expressinghuman CD40 on their surface was investigated by flow cytometry asfollows:

Antibodies from Example 1 were tested for binding to human cell linesexpressing human CD40 on their surface. Protein A purified human mAbs3C3, 3G5, 1B4, 3B6, and 6H6 were incubated with, Raji and Ramos cellsexpressing human CD40 at room temperature on a plate shaker. After 20minutes, the cells were washed with PBS containing 0.1% BSA and 0.05%NaN₃ (PBA) and the bound antibodies were detected by incubating thecells with a PE labeled goat anti-human IgG Fc-specific probe. Theexcess probe was washed from the cells with PBA and the cell associatedfluorescence was determined by analysis using a FACSCanto II™ instrument(BD Biosciences, NJ, USA) according to the manufacturer's directions.

As shown in FIG. 5 (binding to Raji cells) and FIG. 6 (binding to Ramoscells), the human mAbs demonstrated high level binding to cellsexpressing human CD40 as a function of antibody concentration.

Example 6

CD95 Induction on Ramos Cells

Ramos cells were incubated overnight at 37° C., 6% CO₂ with 2 ug/mL ofthe human anti-CD40 mAbs from Example 1. Then next day, they were washedonce with PBA and stained with PE-conjugated anti-CD95 antibody (BectonDickinson®) for 20 minutes at room temperature, with shaking. The excesslabeled antibody was washed off and the samples read on a FACSCanto II™instrument (BD Biosciences®, NJ, USA). As shown in FIGS. 7A and B ((inwhich the shaded plots represent untreated/control cells and the blacklines represent cells treated with the antibodies as indicated), the 3C3and the 1B5-NK antibodies show increases in CD95 and the otherantibodies 3G5, 1B4, 3B6, 6H6, 2E1.2, and 3B6-NS were able to induce astrong increase in expression of surface expressed CD95.

Example 7

Dendritic Cell Activation

Dendritic cells were derived from human monocytes as follows: PMBC'swere added to a T175 cm² flasks and monocytes allowed to adhere for ˜2hours at 37° C., 6% CO₂. The cells were removed and the monocytescultured for 7 days in RPMI containing 10% FBS, 10 ng/mL IL-4 (R&DSystems®) and 100 ng/mL GM-CSF (R&D Systems). The cells were harvestedand confirmed to be dendritic cells by expression of CD11c (not shown).

The cells were then incubated in the presence of 10 ug/mL 3C3 and 3G5human anti-CD40 antibodies from Example land appropriate controls at 37°C., 6% CO₂. After 72 hours, the cells were harvested and the supernatantwas collected and stored for cytokine analysis. The cells were stainedwith the following labeled antibodies for 20 minutes at roomtemperature, shaking: HLA-DR V450, CD54 PE, CD86 APC, and CD83 BV510(all from BD). Cells were then washed twice and analyzed on a FACSCantoII™ instrument (BD Biosciences, NJ, USA). FIG. 8A shows the level ofexpression for each of these markers when incubated with the indicatedantibody or control.

Induction of IL-12p40 was evaluated in the supernatants from these 72hour cultures by ELISA (R&D Systems). FIG. 9A shows the increase inIL-12p40 production with the 3C3 and 3G5 anti-CD40 antibodies relativeto controls as indicated.

In a further experiment cells were incubated in the presence of 10, 1and 0.1 ug/mL 3C3 and 3G5 human anti-CD40 antibodies from Example 1 andappropriate controls at 37° C., 6% CO₂. After 48 hours, the cells wereharvested and the supernatant was collected and stored for cytokineanalysis. The cells were stained with CD54 labeled antibody (BD) for 20minutes at room temperature, shaking. Cells were then washed twice andanalyzed on a FACSCanto II™ instrument (BD Biosciences, NJ, USA). FIG.8B shows the level of expression for CD54 when incubated with theindicated antibody or control.

Induction of IL-12p40 was evaluated in the supernatants from these 48hour cultures by ELISA (R&D Systems). FIG. 9B shows the increase inIL-12p40 production with the 3C3 and 3G5 anti-CD40 antibodies relativeto controls as indicated.

Example 8

B Cell Activation

Whole blood was incubated with 10 ug/mL of 3C3 and 3G5 anti-CD40antibodies from Example 1 overnight at 37° C., 6% CO2. The next day, thefollowing labeled antibodies were used to stain B cells and activationmarkers: CD54 PE, HLA-DR V450, CD23 PerCP-Cy5.5, CD69 APC, CD86 APC,CD38 PerCP-Cy5.5 and CD71 PE. The cells were stained for 20 minutes atroom temperature, shaking, then washed twice and read on a FACSCanto II™instrument (BD Biosciences, NJ, USA). FIG. 10A shows the change in levelof expression on each of these markers relative to controls asindicated.

In a further experiment whole blood was incubated with 10, 1, and 0.1ug/mL of 3C3 and 3G5 anti-CD40 antibodies from Example 1 overnight at37° C., 6% CO₂. The next 5 day, the following labeled antibodies wereused to stain B cells and activation markers: CD19 V500, HLA-DR V450,CD86 APC (all from BD). The cells were stained for 20 minutes at roomtemperature, shaking, then washed twice and read on a FACSCanto II™instrument (BD Biosciences, NJ, USA). FIG. 10B shows the change in levelof expression on each of these markers relative to controls asindicated.

Example 9

NFκB Activation

A luciferase reporter cell line expressing CD40 was incubated for 6hours at 37° C., 6% CO₂ with various concentrations of the humananti-CD40 antibodies from Example 1. Luciferase expression was detectedwith the Luciferase Assay System by Promega® according to themanufacturer's guidelines. FIGS. 11A and 11B show the high level of NFkBactivation induced by 3C3, 3G5, 1B4, 3B6, 6H6, 2E1.2, 1B5-NK, and 3B6-NSantibodies as a function of antibody concentration.

Example 10

Tumor Killing in Raji Xenograft SCID Mouse Model

CB.17 SCID mice (purchased from Taconic Biosciences, Inc.) weremaintained in a pathogen-free mouse facility. Lymphoma Raji cells(1×10⁶) were subcutaneously injected into SCID mice, 5 mice per group.On day 1, 5 and 11, these mice were treated with CD40 human mAbs clone3C3 and 3G5 via intraperitoneal administration, 0.3 mg per dose. Tumorgrowth was measured with calipers 2 times a week. Results of tumorgrowth and survival analysis are shown in FIG. 12, from which it can beseen that, in the tumor challenged mice, treatment with the anti-CD40antibodies inhibited the growth of tumors and significantly prolongedsurvival relative to saline treated controls.

Example 11

Tumor Killing in Ramos Xenograft SCID Mouse Model

CB.17 SCID mice (purchased from Taconic Biosciences, Inc.) weremaintained in a pathogen-free mouse facility. Human lymphoma Ramos cells(1×10⁶) were subcutaneously injected into SCID mice on day 0, 5 mice pergroup. On day 1, 5 and 11, these mice were treated with anti-CD40 humanmAb 3C3 or 3G5 via intraperitoneal administration, 0.3 mg per dose.Tumor growth was measured with calipers 2 times a week.

The results, shown in FIG. 13, indicate that the anti-CD40 mAbssignificantly inhibited the growth in tumor volume compared to salinetreated controls, resulting in the survival of 100% (3G5) or 80% (3C3)of the tumor challenged mice.

Example 12

T-Cell Proliferation

Human Peripheral Blood Mononuclear Cells (PBMCs) isolated from buffycoat preparations were labeled with 0.5 uM carboxyfluoresceinsuccinimidyl ester (CFSE) at room temperature while rotating for 5minutes. The CFSE labeled PBMCs (1.5×10⁶) were dispensed into wells drycoated with anti-CD3 antibody (OKT3) at 0.2 ug/mL.

The CD40 antibodies (3G5, 3C3, 1412) or the isotype control (IgG2) weredispensed into the wells in soluble form at a final concentration of 10ug/mL. The plates were incubated at 37° C. (5% CO₂) On day 6, the cellswere harvested and stained with either anti-CD3-APC or the isotypecontrol and analyzed by flow cytometry. Representative plots are shownin FIG. 14A from which it can be seen that the antibodies significantlyenhanced T-cell proliferation as evidenced by the reduced intensities ofCFSE staining in the CD3+ gate. Results from a repeat experiment areshown in FIG. 14B which shows the increase in dividing cells with theanti-CD40 antibodies relative to the isotype control.

Example 13

Binding to CD40 Independent of Fc Receptor Interaction

Microtiter plates were coated with recombinant human CD40-Fc in PBS, andthen blocked with 5% bovine serum albumin in PBS. Protein A purifiedhuman mAbs (whole IgG and F(ab′)2 fragments as indicated) were added atvarious concentrations and incubated at 37° C. The plates were washedwith PBS/Tween and then incubated with a goat-anti-human IgGF(ab′)2-specific polyclonal reagent conjugated to horseradish peroxidaseat 37° C. After washing, the plates were developed with HRP substrate,and analyzed at OD 450-650 using a microtiter plate reader. Results areshown in FIG. 15. The IgG2 and F(ab)′2 versions of each antibody show asimilar concentration dependence for binding to CD40-Fc.

Example 14

CD40 Activation Independent of Fc Receptor Interaction

The luciferase reporter cell line expressing CD40 from Example 9 abovewas incubated for 6 hours at 37° C., 6% CO₂ with various concentrationsof the human anti-CD40 antibodies (both whole IgG and F(ab′)2 fragmentsas indicated). Luciferase expression was detected with the PromegaLuciferase Assay System according to the manufacturer's guidelines.Results are shown in FIG. 16. These show that binding to the Fc receptoris not required for CD40 mediated activation of the reporter cell lineby 3C3 and 3G5 because intact antibodies with Fc domains and theircorresponding F(ab)′2 versions lacking Fc domains are both able toactivate NFkB in the reporter cell line.

Example 15

CD95 Induction Independent of Fc Receptor Interaction

Ramos cells were incubated overnight at 37° C., 6% CO₂ with variousconcentrations of the human anti-CD40 mAb's, (both whole IgG and F(ab′)2fragments as indicated). The next day they were washed once with PBA andstained with PE-conjugated anti-CD95 antibody (Becton Dickinson) for 20minutes at room temperature with shaking. The excess labeled antibodywas washed off and the samples read a FACSCanto II™ instrument (BDBiosciences, NJ, USA). Results are shown in FIG. 17. These data indicatethat Fc receptor interactions are not required by 3G5 to induce theexpression of CD95 on the CD40+ human lymphoblastoid line Ramos.

Example 16

Synergy with sCD40L

Ramos cells were incubated overnight with the antibody 3C3 plus or minus0.1 mg/ml soluble CD40 Ligand. The cells were then stained withanti-CD95-PE antibody and analyzed by flow cytometry. Results are shownin FIG. 19 and indicate that the anti-CD40 antibody 3C3 actedsynergistically with sCD40L. Accordingly, antibody 3C3 (and anti-CD40antibodies which bind to the same epitope as 3C3) exhibit synergisticagnostic effects with soluble CD40 ligand (sCD40L) and, therefore, havethe ability to synergize with other therapeutic agents, including thosewhich bind to the ligand binding site of human CD40. Representativesynergistic effects include, for example, upregulation of immunefunction (e.g. T cell mediated immune responses as in vaccine therapies,NK activation in cancer therapies), inhibition of cell growth (e.g., incancer therapy), and/or enhanced processing and presentation of anantigen by APCs (e.g., in vaccine therapy).

Example 17

Epitope Mapping of Anti-CD40 Human Antibodies 3C3 and 3G5 and sCD40

i) Generation of Truncated and Mutated Fragments of Soluble CD40(sCD40).

Soluble CD40 (sCD40) cDNA encoding the full length extra cellular domain(ECD) spanning amino acid residues 1-173 (SEQ ID NO: 133), as well asthree smaller fragments coding amino acids 1-94, 36-130 and 84-173, weresynthesized by GenScript and inserted in-frame into a mammalianexpression vector with an N-terminal human kappa light chain and aC-terminal Flag tag. The resulting kappa-sCD40-Flag fusion proteins wereexpressed by transient transfection into ExpiCHO-S cells (SAFC). Sincethe CD40 antibody 3C3 recognizes human and monkey but not mouse CD40, aseries of mutated sCD40aa 1-94 cDNA were designed based on thedifferences between the human and mouse sequences, as shown in thealignments in FIGS. 20 and 21. The mutants were synthesized and clonedby GenScript®. All these truncated or mutated fragments were cloned intothe same vector and expressed by the same cell line as aforementioned.

ii) Determining Binding by ELISA

The binding of 3C3 to the series of sCD40 fragments was tested by ELISA.1 μg/ml of purified kappa-sCD40-Flag fusion proteins or CHO cellsupernatants containing the sCD40 fusion proteins were captured tomicrotiter plates that were pre-coated with 5 ug/ml mouse anti-Flagantibody (Sigma) in PBS and blocked with 5% bovine serum albumin in PBS.Following incubation with the CD40 antibody, the microplates were washedwith PBS/Tween, and incubated with a goat anti-human IgG Fc polyclonalreagent conjugated to horseradish peroxidase. After washing, the plateswere developed with HRP substrate, and analyzed at OD 450-650 using amicrotiter plate reader. An ELISA with a goat anti-human IgG Fab2-HRP tomeasure kappa chain binding was carried out in parallel to validate thesCD40 fusion protein expression from different transfections.

ELISA analysis with ˜1 ug/ml full length sCD40 and the 3 truncatedfragments determined that sCD40 N-terminal residues 1-94 are essentialand sufficient for the binding of 3C3, since the fragment encoding aminoacid residues 1-94 bound to 3C3 as well as the entire ECD, but thefragments encoding amino acid residues 36-130 or 84-173 of this sequencedid not bind at all (see Table 4).

TABLE 4 Fragment amino acid Average OD residues 3C3 αFab2HRP  1-1731.264 1.264  1-94 1.803 1.720 36-130 0.024 1.695 84-173 0.024 1.669Mutant fragment of amino acids 1-94 A (1-5) 0.189 1.718 B (13-15) 2.0321.730 C (25, 26, 28, 30) 1.487 1.685 D (33-36) 0.092 1.631

Based on these results, the critical recognition sites for 3C3 arewithin amino acids 1-35.

To further identify critical regions and amino acid residues for theconformational organization of the binding site of 3C3, ˜2 ug/ml 13mutated sCD40 (amino acid residues 1-94) fragments (4 regional multiplemutations and 9 single mutations) were tested by ELISA (see Tables 5 and6 showing results from separate experiments, and FIG. 22).

TABLE 5 Average OD Fragment amino acid residues 3C3 αFab2HRP 1-94 2.1571.473 Mutant fragment of amino acids 1-94 A (1-5) 0.167 1.489 D (33-36)0.124 1.429 Point Mutation E1G 1.965 1.487 P2Q 2.077 1.490 P3S 2.0111.489 T4V 2.152 1.519 A5T 1.126 1.517 E33A 1.620 1.521 F34L 1.883 1.500T35E 2.072 1.487 E36K 1.369 1.433 PBA 0.031 0.011

TABLE 6 OD 3C3 3G5 αFab2HRP Fragment amino acid residues Full length1-173 2.364 2.214 1.525  1-94 2.151 2.170 1.755 36-130 0.029 0.048 1.71684-173 0.024 0.038 1.599 Mutant fragment of amino acids 1-94 A (1-5)0.250 2.139 1.699 B (13-15) 2.375 1.876 1.710 C (25, 26, 28, 30) 2.0162.161 1.604 D (33-36) 0.233 0.042 1.548 Point Mutation E1G 2.011 2.0831.720 P2Q 2.197 2.158 1.754 P3S 2.012 2.188 1.712 T4V 2.213 2.210 1.664AST 1.511 2.201 1.698 E33A 1.695 0.074 1.709 F34L 1.845 1.192 1.686 T35E2.102 2.128 1.682 E36K 1.689 1.930 1.674 <0.25 0.25 < x < 1.2 1.2 < x <1.9

Multiple mutations of residues 1-S almost completely abrogated 3C3.Point mutations of residues 1-4 did not reduce binding to 3C3. The pointmutation of residue 5 dramatically reduced binding but not to the extentof the multiple mutation protein.

Multiple mutations of residues 13-15 did not reduce 3C3 binding.Multiple mutations of residues 25, 26, 28 and 30 caused a slightreduction in 3C3. Point mutations were not tested in these regions.

Multiple mutations of residues 33-36 almost completely abrogated 3C3binding. The point mutation of residue 35 had no effect on binding. Thepoint mutations of 33, 34 and 36 decreased 3C3 binding but not to theextent of the multiple mutation protein. An alternate CD40 antibody,3G5, was tested for binding to all fragments and mutants and was shownto be different than 3C3 (Table 6). The multiple mutation of residues1-5 did not reduce binding while the mutation of residues 33-36eliminated binding. Unlike 3C3, the point mutation of residue 33completely eliminated binding and the mutation of 34 significantlyreduced binding.

Example 18

Biological and Toxicity Profile

A non-GLP pilot study was performed in naive cynomolgus macaques. Thisstudy was designed to provide preliminary data on the biological andtoxicity profile of 3C3. An alternative anti-CD40 antibody (3G5) wasalso evaluated. The test articles were administered by intravenousinjection in a saphenous vein on Day 1 (0.2 mg/kg or vehicle) and againon Day 29 (2 mg/kg or vehicle). Animals also received a subcutaneous (1mg) injection of keyhole limpet hemocyanin (KLH) on Day 1 and 29.Evaluations for potential test article-related effects were based onclinical signs, body temperature, clinical pathology parameters(hematology, coagulation, clinical chemistry, and urinalysis), anti-drugantibodies, cytokines, T-cell dependent antibody response analyses(TDAR), flow cytometry, and toxicokinetic parameters. Body weights wererecorded once prior to test article administration and weeklythereafter. This was designed as a survival study with no plannednecropsy.

Administration of 3C3 or 3G5 in this study was well tolerated incynomolgus monkeys without any toxicity parameter being significantlyoutside of control levels. Of note was the minimal elevations ofseparate aminotransferase (AST), alanine aminotransferase (ALT) andcreatinine kinase in monkeys dosed with 3C3 (FIGS. 23A-23C).Pharmacologic decreases in IL-12 (FIG. 24) white blood cells (FIG. 25A),neutrophils (FIG. 25B) and lymphocytes (FIG. 25C), were seen in bothanti-CD40 dosed animals, with most significantly a transient decrease inB cells (FIGS. 26 and 27). In conclusion, 3C3 and 3G5 under theconditions of this showed minimal evidence of toxicity.

Example 19

B Cell Proliferation Independent of Fc Interaction

Human B cells were isolated from peripheral blood mononuclear cells bymagnetic selection using CD19 beads. The cells were labeled with 0.5 uMcarboxyfluorescein succinimidyl ester (CFSE) at room temperature whilerotating for 5 minutes. The labeled cells were cultured in the presenceof either the anti-CD40 mAb 3C3 or an isotype control (both whole IgGand F(ab′)2 fragments) for 6 days. Cells were then harvested andanalyzed by flow cytometry for proliferation. The results are shown inFIG. 28 and indicate that binding to the Fc receptor is not required forCD40 mediated proliferation with 3C3 because intact antibodies with Fcdomains and their corresponding F(ab)′2 versions lacking Fc domains areboth able to induce proliferation of B cells.

Example 20

Synergy with CD40L in Human B Cells

Human B cells were isolated and labeled as in Example 19. The anti-CD40mAb 3C3 or an isotype control at 0.1 ug/mL were incubated with the cellsfor 6 days in the presence or absence of 0.1 ug/mL soluble CD40L(Immunex®). FIG. 29 shows that no significant proliferation is observedwith either the 3C3 alone or the isotype control antibody combined withCD40L, however proliferation is induced when CD40L is combined with 3C3in the culture.

Dendritic cells were prepared and cultured with 0.5 ug/mL of 3C3 as inExample 7 either with or without 0.1 ug/mL soluble CD40L added. IL-12p40production was measured by ELISA (R&D Systems). FIG. 30 shows thatrelative to the low level of production by 3C3 alone or the isotypecontrol with CD40L, the combination of 3C3 and CD40L induced higherlevels of IL-12p40.

Example 21

Cytokine Response in Whole Blood

Whole blood was incubated overnight with 10 ug/mL isotype control or3C3, or LPS as a positive control. Next day, the plasma was collectedand cytokines measured by ELISA (R&D Systems). The results are shown inFIG. 31 and indicate no significant production of inflammatorycytokines.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

SUMMARY OF SEQUENCE LISTING SEQ ID NO: DESCRIPTION   1Human CD40 (GenBank Accession No.: P25942)MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSL CQPGQKLVSD CTEFTETECLPCGESEFLDT WNRETHCHQH KYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCVLHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEK CHPWTSCETK DLVVQQAGTNKTDVVCGPQD RLRALVVIPI IFGILFAILL VLVFIKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ   2Human CD40L (GenBank Accession No.: NP_000065)MIETYNQTSP RSAATGLPIS MKIFMYLLTV FLITQMIGSA LFAVYLHRRL DKIEDERNLHEDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ FEGFVKDIML NKEETKKENS FEMQKGDQNPQIAAHVISEA SSKTTSVLQW AEKGYYTMSN NLVTLENGKQ LTVKRQGLYY IYAQVTFCSNREASSQAPFI ASLCLKSPGR FERILLRAAN THSSAKPCGQ QSIHLGGVFE LQPGASVFVNVTDPSQVSHG TGFTSFGLLK   3 3G5-VHQVQLVESGGGVVQPGKSLRLSCAASGFTFSSNGIHWVRQAPGKGLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARASGSGSYYNFFDWGQGTLVTVSS   4 3G5-VLEIVMTQSPATLSVSPGERATLSCRASQSVRSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTINSLQSEDFAVYYCQQHNKWITFGQGTRLEIK   5 3G5-VH CDR1 (KABAT) SNGIH  6 3G5-VH CDR1 (CHOTHIA) GFTFSSN   7 3G5-VH CDR2 (KABAT)VIWSDGSNKFYADSVKG   8 3G5-VH CDR2 (CHOTHIA) WSDGSN   93G5-VH CDR3 (KABAT) ASGSGSYYNFFDY  10 3G5-VH CDR3 (CHOTHIA)ASGSGSYYNFFDY  11 3G5-VL CDR1 (KABAT) RASQSVRSNLA  123G5-VL CDR1 (CHOTHIA) RASQSVRSNLA  13 3G5-VL CDR2 (KABAT) GASTRAT  143G5-VL CDR2 (CHOTHIA) GASTRAT  15 3G5-VL CDR3 (KABAT) QQHNKWIT  163G5-VL CDR3 (CHOTHIA) QQHNKWIT  17 3C3-VHQVQLVESGGGVVQPGRSLRLSCAGSGFIFSRYGMYWVRQAPGKGLEWVAVIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARESPWYYFDYWGQGTLVTVSS  18 3C3-VLDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYKSAPFTFGPGTKVDIK  19 3C3-VH CDR1 (KABAT) RYGMY 20 3C3-VH CDR1 (CHOTHIA) GFIFSRY  21 3C3-VH CDR2 (KABAT)VIWYDGSYKYYADSVKG  22 3C3-VH CDR2 (CHOTHIA) WYDGSY  233C3-VH CDR3 (KABAT) ESPWYYFDY  24 3C3-VH CDR3 (CHOTHIA) ESPWYYFDY  253C3-VL CDR1 (KABAT) RASQGISNYLA  26 3C3-VL CDR1 (CHOTHIA) RASQGISNYLA 27 3C3-VL CDR2 (KABAT) AASTLQS  28 3C3-VL CDR2 (CHOTHIA) AASTLQS  293C3-VL CDR3 (KABAT) QKYKSAPFT  30 3C3-VL CDR3 (CHOTHIA) QKYKSAPFT  313B6-VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGTGGSTYYADSVKGRFTISRDNSKNTLYVQMNSLRAEDTAVYYCAKRAGGSFYYYYGMDVWGQGTTVTVSS  32 3B6-VLYLGSNRASGVPDRFNGSGSGTDFTLKISRVEAEDFGVYYCMQALQTPWTFGHGTKVEIK  333B6-VH CDR1 (KABAT) SYAMS  34 3B6-VH CDR1 (CHOTHIA) GFTFSSY  353B6-VH CDR2 (KABAT) GITGTGGSTYYADSVKG  36 3B6-VH CDR2 (CHOTHIA) TGTGGS 37 3B6-VH CDR3 (KABAT) RAGGSFYYYYGMDV  38 3B6-VH CDR3 (CHOTHIA)RAGGSFYYYYGMDV  39 3B6-VL CDR1 (KABAT) RSSQSLLHSTGYNYLD  403B6-VL CDR1 (CHOTHIA) RSSQSLLHSTGYNYLD  41 3B6-VL CDR2 (KABAT) LGSNRAS 42 3B6-VL CDR2 (CHOTHIA) LGSNRAS  43 3B6-VL CDR3 (KABAT) MQALQTPWT  443B6-VL CDR3 (CHOTHIA) MQALQTPWT  45 6H6-VHQVQLVESGGGVVQPGRSLRFSCAASGFTLSSYGMHWVRQAPGKGLEWVAVIWDDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGGSGRYYNYFDYWGQGTLVTVSS  46 6H6-VLEIVMTQSPATLSVSPGERATLSCRASQSVRSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQSEDFAVYYCQQHNNWLTFGGGTKVEIK  47 6H6-VH CDR1 (KABAT) SYGMH 48 6H6-VH CDR1 (CHOTHIA) GFTLSSY  49 6H6-VH CDR2 (KABAT)VIWDDGSNKYYADSVKG  50 6H6-VH CDR2 (CHOTHIA) WDDGSN  516H6-VH CDR3 (KABAT) AGGSGRYYNYFDY  52 6H6-VH CDR3 (CHOTHIA)AGGSGRYYNYFDY  53 6H6-VL CDR1 (KABAT) RASQSVRSNLA  546H6-VL CDR1 (CHOTHIA) RASQSVRSNLA  55 6H6-VL CDR2 (KABAT) GASTRAT  566H6-VL CDR2 (CHOTHIA) GASTRAT  57 6H6-VL CDR3 (KABAT) QQHNNWLT  586H6-VL CDR3 (CHOTHIA) QQHNNWLT  59 1B4-VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVRQVPGKGLEWVSGITGSGANTFYTDSVKGRFTISRDNSNNSLYLQMNSLRADDTAVYYCAKRNGGSYYYYYGMDVWGQGTTVTVSS  60 1B4-VLDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSSGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQIPWTFGQGTKVEIK  61 1B4-VH CDR1 (KABAT)SYAMT  62 1B4-VH CDR1 (CHOTHIA) GFTFSSY  63 1B4-VH CDR2 (KABAT)GITGSGANTFYTDSVKG  64 1B4-VH CDR2 (CHOTHIA) TGSGAN  651B4-VH CDR3 (KABAT) RNGGSYYYYYGMDV  66 1B4-VH CDR3 (CHOTHIA)RNGGSYYYYYGMDV  67 1B4-VL CDR1 (KABAT) RSSQSLLHSSGYNYLD  681B4-VL CDR1 (CHOTHIA) RSSQSLLHSSGYNYLD  69 1B4-VL CDR2 (KABAT) LGSNRAS 70 1B4-VL CDR2 (CHOTHIA) LGSNRAS  71 1B4-VL CDR3 (KABAT) MQALQIPWT  721B4-VL CDR3 (CHOTHIA) MQALQIPWT  73 3B6-NS-VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGTGGSTYYADSVKGRFTISRDNSKNTLYVQMNSLRAEDTAVYYCAKRAGGSFYYYYGMDVWGQGTTVTVSS  74 3B6-NS-VLDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSTGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDFGVYYCMQALQTPWTFGHGTKVEIK  753B6-NS-VH CDR1 (KABAT) SYAMS  76 3B6-NS-VH CDR1 (CHOTHIA) GFTFSSY  773B6-NS-VH CDR2 (KABAT) GITGTGGSTYYADSVKG  78 3B6-NS-VH CDR2 (CHOTHIA)TGTGGS  79 3B6-NS-VH CDR3 (KABAT) RAGGSFYYYYGMDV  803B6-NS-VH CDR3 (CHOTHIA) RAGGSFYYYYGMDV  81 3B6-NS-VL CDR1 (KABAT)RSSQSLLHSTGYNYLD  82 3B6-NS-VL CDR1 (CHOTHIA) RSSQSLLHSTGYNYLD  833B6-NS-VL CDR2 (KABAT) LGSNRAS  84 3B6-NS-VL CDR2 (CHOTHIA) LGSNRAS  853B6-NS-VL CDR3 (KABAT) MQALQTPWT  86 3B6-NS-VL CDR3 (CHOTHIA) MQALQTPWT 87 2E1.2-VHQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWDDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGSSGRYYNYFDYWGQGTLVTVSS  88 2E1.2-VL2EIVMTQSPATLSVSPGERATLSCRASQSVRSNLAWYQQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTEFTLTISSLQSEDFAVYHCQQYNKWLIFGGGTKVEIK  89 2E1.2-VH CDR1 (KABAT)SYGMH  90 2E1.2-VH CDR1 (CHOTHIA) GFTFSSY  91 2E1.2-VH CDR2 (KABAT)VIWDDGSNKYYADSVKG  92 2E1.2-VH CDR2 (CHOTHIA) WDDGSN  932E1.2-VH CDR3 (KABAT) AGSSGRYYNYFDY  94 2E1.2-VH CDR3 (CHOTHIA)AGSSGRYYNYFDY  95 2E1.2-VL2 CDR1 (KABAT) RASQSVRSNLA  962E1.2-VL2 CDR1 (CHOTHIA) RASQSVRSNLA  97 2E1.2-VL2 CDR2 (KABAT) GASTRAT 98 2E1.2-VL2 CDR2 (CHOTHIA) GASTRAT  99 2E1.2-VL2 CDR3 (KABAT) QQYNKWLI100 2E1.2-VL2 CDR3 (CHOTHIA) QQYNKWLI 101 1B5-NK-VHQVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMEIWVRQAPGKGLEWVTLAVAGWYFDFWGRGT LVTVSS102 1B5-NK-VLDIQMTQSPSSLSASVGDRVTITCRASQGVRKYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYFSAPYTFGQGTKLEIK 103 1B5-NK-VH CDR1 (KABAT)SFGMH 104 1B5-NK-VH CDR1 (CHOTHIA) GFTFSSF 105 1B5-NK-VH CDR2 (KABAT)LIWFDGSSKYYADSVKG 106 1B5-NK-VH CDR2 (CHOTHIA) WFDGSS 1071B5-NK-VH CDR3 (KABAT) GFAAVAGWYFDF 108 1B5-NK-VH CDR3 (CHOTHIA)GFAAVAGWYFDF 109 1B5-NK-VL CDR1 (KABAT) RASQGVRKYLA 1101B5-NK-VL CDR1 (CHOTHIA) RASQGVRKYLA 111 1B5-NK-VL CDR2 (KABAT) AASTLQS112 1B5-NK-VL CDR2 (CHOTHIA) AASTLQS 113 1B5-NK-VL CDR3 (KABAT)QKYFSAPYT 114 1B5-NK-VL CDR3 (CHOTHIA) QKYFSAPYT 1153G5 VH with leader sequence underlinedAtggagtttgggctgacctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagttggtggaatctGggggaggcgtggtccagcctgggaagtccctgagactctcctgtgcagcgtctggattcaccttcagtagcaatgGcattcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatctggtctgatggaagtaataaAttctatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctatatctgcaaatgaAcagcctgagagccgaggacacggctgtatattactgtgcgagagcctctggttcggggagttattataacttctttgactactggggccagggaaccctggtcaccgtctcctca 1163G5 VL with leader sequence underlineAtggaagccccagcgcagcttctcttcctcctgctactctggctcccagatagcactggagaaatagtgatgacgcagTctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagaagtaacTtagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccaccagggccactggtatccCagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcaacagcctgcagtctgaagattttgcagtttattactgtcagcagcataataagtggatcaccttcggccaagggacacgactggagattaaa 1173C3 VH with leader sequence underlinedAtggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctggGggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagggtctggattcattttcagtcgctatggcatgTactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagttataaatactatGcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgAgagccgaggacacggctgtgtattactgtgcgagagaatcaccatggtactactttgactactggggccagggaaccctggtcaccgtctcctct 118 3C3 VL with leader sequence underlinedAtggacatgagggtccctgctcagctcctgggactcctgctgctctggctcccagataccagatgtgacatccagatgacCcagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcgagtcagggcattagcaattaTttagcctggtatcagcagaaaccagggaaagttcctaagctcctgatctatgctgcatccactttgcaatcaggggtcccAtctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcaaaagtataagagtgccccattcactttcggccctgggaccaaagtggatatcaaa 1193B6 VH with leader sequence underlinedAtggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgtgaggtgcagctgttggagtctggggGaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctGggtccgccaggctccagggaaggggctggagtgggtctcaggtataactggtactggtggtagcacatactacgcagActccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatgtgcaaatgaacagcctgagagcCgaggacacggccgtatattactgtgcgaaaagggctggtgggagcttctactactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca 1203B6 VL with leader sequence underlinedAtgaggctccctgctcagctcctggggctgctaatgctctgggtctctggatccagtggggatattgtgatgactcagtctcCactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagcctcctgcatagtactggataCaactatttggattggtacctgcagaagccagggcagtctccacagctcctgatctatttgggttctaatcgggcctccgggGtccctgacaggttcaatggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggattttggggtttattactgcatgcaagctctacaaactccgtggacgttcggccacgggaccaaggtggaa atcaaa121 6H6 VH with leader sequence underlinedAtggagtttgggctgagctgggtattcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctggggGaggcgtggtccagcctgggaggtccctgagattctcctgtgcagcgtctggattcaccctcagtagctatggcatgcactgGgtccgccaggctccaggcaaggggctggagtgggtggcagttatatgggatgatggaagtaataaatactatgcagactCcgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggAcacggctgtctattactgtgcgagagcggggggttcggggaggtattataactactttgactactggggccagggaaccctggtcaccgtctcctca 1226H6 VL with leader sequence underlinedAtggaagccccagcgcagcttctcttcctcctgctactctggctcccagataccactggagaaatagtgatgacgcagtctccAgccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagaagcaacttagcctggTaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccaccagggccactggtatcccagccaggttcagTggcagtgggtctgggacagacttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcagcaTaataactggctcactttcggcggagggaccaaggtggagatcaaa 1231B4 VH with leader sequence underlinedAtggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccaatgtgaggtgcagctgttggaatctgggggaGgcttggtacagcctggggggtccctgagactctcctgtgcggcctctgggttcacctttagcagctatgccatgacctgggtcCgccaggttccagggaagggcctggagtgggtctcaggtattactggtagtggtgctaacacattctacacagactccgtgaAgggccggttcaccatttccagagacaattccaataattcgctgtatctgcaaatgaacagcctgagagccgatgacacggcCgtatactactgtgcgaaaagaaatggtgggagttactactactactacggcatggacgtctggggccaagggaccacggtcaccgtgtcctca 1241B4 VL with leader sequence underlinedAtgaggctccctgctcagctcctggggctgctaatgctctgggtctctggatccagtggggatattgtgatgactcagtctccacTctccctgcccgtcacccctggagagccggcctccatctcctgcaggtcaagtcagagcctcctgcatagtagtggatacaactaTttggattggtacctgcagaagccagggcagtctccacaactcctgatctatttgggttctaatcgggcctccggggtccctgacAggttcagtggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaagctctacaaattccgtggacgttcggccaagggaccaaggtggaa atcaaa125 2E1.2 VH with leader sequence underlinedAtggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgggggaggcgtggtCcagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaAggggctggagtgggtggcagttatatgggatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagAcaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagcgggaagttcggggaggtattataactactttgactactggggccagggaaccctggtcaccgtctcctca 1262E1.2 VL2 with leader sequence underlinedAtggaagccccagcgcagcttctcttcctcctgctactctggctcccagataccactggagaaatagtgatgacgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttaggagcaacttagcctggtatcagcagaaacctggccaggctcccaggctcctcatctatggtgcatccaccagggccactggtatcccagacaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttatcactgtcagcagtataataagtggctcattttcggcggagggaccaaggtggagatcaaa 1271B5 VH with leader sequence underlinedAtggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgggggaggcgtggtccagcCtgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctttggcatgcactgggtccgccaggctccaggcaaggggctggaGtgggtgacacttatatggtttgatggaagttctaaatactatgcagactccgtgaagggccgattcaccatctccagagacaactccaacaacacGctgtatctgcaaatgaacagcctgagagccgaggacacggctgtatattactgtgtgagaggttttgcagcagtggctgggtggtacttcgatttctggggccgtggcaccctggtcactgtctcctca 1281B5 VL with leader sequence underlinedAtggacatgagggtccctgctcagctcctgggactcctgctgctctggctcccagataccagatgtgacatccagatgacccagtctccatcctcccTgtctgcatctgtaggagacagagtcaccatcacttgccgggcgagtcagggcgttagaaagtatttagcctggtatcagcagaaaccagggaaAgttcctaagctcctgatctatgctgcatccactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccaTcagcagcctgcagcctgaagatgttgcaacttattactgtcaaaagtatttcagtgccccgtacacttttggccaggggaccaaactggagatcaaa 1293B6-NS VH with leader sequence underlinedAtggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgtgaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaggtataactggtactggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatgtgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaaaagggctggtgggagcttctactactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca 1303B6-NS VL with leader sequence underlinedatgaggctccctgctcagctcctggggctgctaatgctctgggtctctggatccagtggggatattgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagcctcctgcatagtactggatacaactatttggattggtacctgcagaagccagggcagtctccacagctcctgatctatttgggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggattttggggtttattactgcatgcaagctctacaaactccgtggacgttcggccacgggaccaaggtggaa atcaaa131 1B5-NK VH with leader sequence underlinedatggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctttggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtgacacttatatggtttgatggaagttctaaatactatgcagactccgtgaagggccgattcaccatctccagagacaactccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtatattactgtgtgagaggttttgcagcagtggctgggtggtacttcgatttctggggccgtggcaccctggtcactgtctcctca 1321B5-NK VL with leader sequence underlinedAtggacatgagggtccctgctcagctcctgggactcctgctgctctggctcccagataccagatgtgacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcgagtcagggcgttagaaagtatttagcctggtatcagcagaaaccagggaaagttcctaagctcctgatctatgctgcatccactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcaaaagtatttcagtgccccgtacactttggccaggggaccaaactggagatcaaa 133Human CD40 Extracellular DomainEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLR 134Immunoglobulin heavy constant gamma 2 (IgHG2) (Uniprot P01859)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 1353C3 heavy chain with variable region in italics and constantdomain in boldQVQLVESGGGVVQPGRSLRLSCAGSGFIFSRYGMYWVRQAPGKGLEWVAVIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARESPWYYDFYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPG 1363C3 light chain with variable region in italics and constantdomain in boldDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYKSAPFTFGPGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 1373C3 heavy chain with leader sequence underlined, variable regionin italics and constant domain in bold MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAGSGFIFSRYGMYWVRQAPGKGLEWVAVIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARESPWYYFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 1383C3 light chain with leader sequence underlined, variable regionin italics and constant domain in bold MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYKSAPFTFGPGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The invention claimed is:
 1. An isolated nucleic acid encoding thevariable region of a light chain, heavy chain, or both light and heavychains of an antibody which binds to human CD40, wherein the heavyand/or light chain variable regions comprise the amino acid sequence setforth in SEQ ID NOs: 17 and 18, respectively.
 2. An expression vectorcomprising the nucleic acid of claim
 1. 3. A cell transformed with anexpression vector of claim
 2. 4. An isolated nucleic acid encoding thevariable region of a light chain, heavy chain, or both light and heavychains of an antibody which binds to human CD40, wherein the heavyand/or light chain variable regions comprise the CDR sequences from theheavy and/or light chain variable regions comprising the amino acidsequences set forth in SEQ ID NOs: 17 and 18, respectively.
 5. Anexpression vector comprising the nucleic acid of claim
 4. 6. A celltransformed with an expression vector of claim
 5. 7. An isolated nucleicacid encoding the variable region of a light chain, heavy chain, or bothlight and heavy chains of an antibody which binds to human CD40, whereinthe heavy chain variable region comprises the CDR1, CDR2, and CDR3sequences set forth in SEQ ID NOs: 19, 21, and 23, respectively, and thelight chain variable region comprises the CDR1, CDR2, and CDR3 sequencesset forth in SEQ ID NOs: 25, 27, and 29, respectively.
 8. An expressionvector comprising the nucleic acid of claim
 7. 9. A cell transformedwith an expression vector of claim
 8. 10. A method of producing anantibody comprising culturing the cell of claim 3, wherein the antibodyis produced.
 11. A method of producing an antibody comprising culturingthe cell of claim 6, wherein the antibody is produced.